Polymyxin derivatives and uses thereof

ABSTRACT

The present invention relates to a polymyxin derivative wherein R1, R2 and R3 are optional and R1, R2, R3, R5, R8 and R9 are cationic or neutral amino acid residues selected so that the total number of positive charges at physiological pH is at least two but no more than three; and to a combination product comprising at least two such derivatives. The invention further relates to a method for treating, alleviating or ameliorating an infection in a subject, caused by a Gram-negative bacterium by administering a therapeutically effective amount of a derivative according to the present invention to said subject; to a method for sensitizing Gram-negative bacteria to an antibacterial agent by administering, simultaneously or sequentially in any order a therapeutically effective amount of said antibacterial agent and a derivative according to the present invention to said subject; to methods for developing novel antibiotics; for reducing the nephrotoxicity, for improving the pharmacokinetic properties of natural polymyxins and octapeptins; and for sensitizing clinically important bacteria to a host defense mechanism complement present in serum. Finally, the invention relates to a process for preparing such polymyxin derivatives.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/891,629, filed Aug. 10, 2007, which claims the benefit of U.S. Ser.No. 60/837,426, filed Aug. 11, 2006, the disclosures of each of whichare incorporated herein by reference in their entireties. The contentsof any patents, patent applications, and references cited throughoutthis specification are hereby incorporated by reference in theirentireties.

TECHNICAL FIELD

The present invention relates to polymyxin derivatives and to usesthereof in the treatment of infections caused by Gram-negative bacteria.The polymyxin derivatives may have antibacterial effects or maysensitize bacteria to enhance the effects of other antibacterial agents.

BACKGROUND

Sepsis kills more than 215,000 Americans each year. It is estimated that750,000 Americans are infected with severe sepsis and 29% of them diefrom it each year. Sepsis deaths make 9% of all death cases in the U.S.Sepsis kills as many Americans as myocardial infections, even more thantraffic accidents.

Two to three million Americans acquire a hospital infection each yearand 10% of these infections progress to sepsis. More than 90,000 ofthese patients die from sepsis infected in hospitals.

Severe sepsis and septic shock (severe sepsis combined with low bloodpressure) took up to 135,000 lives each year in the intensive care units(ICU) in the European Union according to the OECD Health Report of 2000.In Britain, 5,000 out of 100,000 patients who acquired a hospitalinfection die from sepsis every year in acute care hospitals belongingto the NHS organisation.

The death toll has increased year after year due to the fact that thenumber of patients predisposed to sepsis, such as the elderly, prematureneonates, and cancer patients, has increased, not least because manyserious illnesses are more treatable than before. Also the use ofinvasive medical devices and aggressive procedures has increased.

Gram-negative bacteria cause more than 40% of all septicemic infectionsand many of the Gram-negative bacteria are extremely multiresistant.Gram-negative bacteria provide a harder challenge in therapy thanGram-positives, as they possess a unique structure, the outer membrane,as their outermost structure. Lipopolysaccharide molecules located onthe outer membrane inhibit the diffusion of many antibacterial agentsdeeper into the cell, where their ultimate targets are located. Morethan 95% of the novel antibacterial agents isolated from nature orchemically synthesized in 1972-1991 lacked activity againstGram-negatives (Vaara 1993).

Polymyxins are a group of closely related antibiotic substances producedby strains of Paenibacillus polymyxa and related organisms. Thesecationic drugs are relatively simple peptides with molecular weights ofabout 1000. Polymyxins, such as polymyxin B, are decapeptideantibiotics, i.e. they are made of ten (10) aminoacyl residues. They arebactericidal and especially effective against Gram-negative bacteriasuch as Escherichia coli and other species of Enterobacteriaceae,Pseudomonas, Acinetobacter baumannii, and others. However, polymyxinshave severe adverse effects, including nephrotoxicity and neurotoxicity.These drugs thus have limited use as therapeutic agents because of highsystemic toxicity.

Polymyxins have been used in the therapy of serious infections caused bythose bacteria, but because of the toxicity, their use was largelyabandoned in the 70's when newer, better tolerated antibiotics weredeveloped. The recent emergence of multiresistant strains ofGram-negative bacteria has necessitated the therapeutic use ofpolymyxins as the last resort, in spite of their toxiticy, and as manyof the less toxic antibiotics have already lost their effectivenessagainst particular strains of the said bacteria, the use of polymyxinshas again increased.

Accordingly, polymyxins have now been recalled to the therapeuticarsenal, although, due to their toxicity, on a very limited scale. Theirsystemic (i.e. non-topical) use is, however, largely restricted to thetherapy of life-threatening infections caused by multiply resistantstrains of Ps. aeruginosa and A. baumannii as well as bycarbapenem-resistant enteric bacteria.

Polymyxins consist of a cyclic heptapeptide part and a linear partconsisting of a tripeptide portion and a hydrophobic fatty acid taillinked to the α-amino group of the N-terminal amino acid residue of thetripeptide and may be represented by the general formula:

wherein R1-R3 represent the tripeptide side chain portion; R4-R10 theheptapeptide ring portion and R(FA) represents the hydrophobic fattyacid tail linked to the α-amino group of the N-terminal amino acidresidue of the tripeptide.

The polymyxin group includes the following polymyxins: A1, A2, B1-B6, C,D1, D2, E1, E2, F, K1, K2, M, P1, P2, S, and T (Storm et al. 1977;Srinivasa and Ramachandran 1979). All polymyxins are polycationic andpossess five (5) positive charges, with the exception of polymyxin D, F,and S which possess four (4) positive charges. It should be noted thatmodified polymyxins that lack the fatty acid part R(FA) but carry R1—R10have one additional positive charge when compared to the naturalpolymyxins they derived from, due to the free α-amino group in theN-terminus of the derivative. Accordingly, for example, such aderivative of polymyxin B or polymyxin E carries six (6) positivecharges in total.

The clinically used polymyxin B and polymyxin E differ from each otheronly in the residue R6, which is D-phenylalanyl residue in polymyxin Band D-leucyl residue in polymyxin E.

Also circulin A and B are classified as polymyxins (Storm et al. 1977).They differ from other polymyxins only in carring isoleucyl residue inthe position R7 whereas other polymyxins have either threonyl or leucylresidue in the said position. For an overview of the structures of somepolymyxins, see Table 1.

TABLE 1 The structure of selected polymyxins and octapeptin as well asselected derivatives thereof Compound R(FA) R1 R2 R3 R4 R5 R6 R7 R8 R9R10 Polymyxin B MO(H)A- Dab- Thr- Dab- *Dab- Dab- D Phe- Leu- Dab Dab*Thr Colistin (polymyxin E) MO(H)A- Dab- Thr- Dab- *Dab- Dab- D Leu-Leu- Dab Dab *Thr Colistin sulphomethate MO(H)A- sm-Dab- Thr- sm-Dab-*Dab- Sm-Dab- D Leu- Leu- sm--Dab- sm--Dab- *Thr Polymyxin A MO(H)A-Dab- Thr- D Dab- *Dab- Dab- D Leu- Thr- Dab Dab *Thr Polymyxin M MOADab- Thr- Dab- *Dab- Dab- D Leu- Thr- Dab Dab *Thr Polymyxin D MO(H)A-Dab- D Ser- Dab- *Dab- Dab- D Leu- Thr- Dab Dab *Thr Circulin A MOA Dab-Thr- Dab- *Dab- Dab- D Leu- Ile- Dab Dab *Thr Octapeptin A OHMDA — —Dab- *Dab- Dab- D Leu- Leu- Dab Dab *Thr Deacylcolistin (DAC) Dab- Thr-Dab- *Dab- Dab- D Leu- Leu- Dab Dab *Thr Polymyxin E nonapeptide (PMEN)Thr- Dab- *Dab- Dab- D Leu- Leu- Dab Dab *Thr Deacylpolymyxin B (DAPB)Dab- Thr- Dab- *Dab- Dab- D Phe- Leu- Dab Dab *Thr Polymyxin Bnonapeptide (PMBN) Thr- Dab- *Dab- Dab- D Phe- Leu- Dab Dab *ThrPolymyxin B octapeptide (PMBO) Dab- *Dab- Dab- D Phe- Leu- Dab Dab *ThrPolymyxin B heptapeptide (PMHP) *Dab- Dab- D Phe- Leu- Dab Dab *Thr

Polymyxin B is represented by the following formula:

Commercially available polymyxin B is a mixture, where R-FA ispredominantly 6-methyloctanoyl (6-MOA, in polymyxin B1) but may also bea related fatty acyl such as 6-methylheptanoyl (6-MHA, in polymyxin B2),octanoyl (in polymyxin B3), or heptanoyl (polymyxin B4) (Sakura et al.2004). All these variants are equally potent against Gram-negatives suchas E. coli (Sakura et al. 2004). Quite analogously, in polymyxin E1(colistin A) and in circulin A the R-FA is 6-MOA and in polymyxin E2(colistin B) and in circulin B the R-FA is 6-MHA. Numerous researchershave attached various hydrophobic moieties including various fatty acylresidues to the N-terminus of polymyxin derivatives and analogues andhave shown that the resulting derivatives have potent antibacterialactivity (Chihara et al. 1973, Sakura et al. 2004 and in US patentpublication 2006004185. Even the derivative that carries the bulkyhydrophobic 9-fluorenylmethoxycarbonyl residue as the R-FA is almost aspotent as polymyxin B in inhibiting the growth of E. coli and otherGram-negative bacteria (Tsubery et al. 2001).

For biological activity the heptapeptide ring structure is essential(Storm et al. 1997). A derivative with an octapeptide ring issignificantly less active as an antibiotic.

Multiple modifications of polymyxins and multiple polymyxin-likesynthetic molecules have been made, and with certain limits they havepreserved their biological activity. The modifications comprise but arenot limited to those in the side chain, as well as molecules in which aninherent hydrophobic amino acid residue (such as DPhe or Leu) has beenreplaced with another hydrophobic amino acid residue or in which thecationic Dab has been replaced with another cationic amino acyl residue,such as Lys, Arg, or ornithine residue (Storm et al. 1997, Tsubery etal. 2000a, Tsubery et al. 2002, US patent publication 2004082505, Sakuraet al. 2004, US patent publication 2006004185).

Other modifications that result in microbiologically at least partiallyactive compounds comprise but are not limited to alkanoyl esters wherethe OH-groups of the threonyl residues form esters with alkanoyls suchas propionyl and butyryl (U.S. Pat. No. 3,450,687).

Octapeptins are otherwise identical to polymyxin E (colistin) but have acovalent bond instead of the residues R1—R2 (Table 1). In thisinvention, the R positions are numbered according to those in thenatural polymyxins and thus the only amino acyl residue in the sidechain of octapeptins is defined as R3. Accordingly, octapeptins areoctapeptides whereas all natural polymyxins are decapeptides, and theypossess only four (4) positive charges. The R-FA residues among variousoctapeptins (A1, A2, A3, B1, B2, B3, C1) include the following:3-OH-8-methyldecanoic acid, 3-OH-8-methylnonanoic acid, andβ-OH-6-methyloctanoic acid. Derivatives that possess a fatty acylresidue with 6 to 18 carbon atoms have a potent antibacterial activityagainst E. coli (Storm et al. 1977).

The first target of polymyxins in Gram-negative bacteria is their outermembrane (OM) that is an effective permeability barrier against manynoxious agents including large (Mw more than 700 d) antibiotics as wellas hydrophobic antibiotics. By binding to the lipopolysaccharide (LPS)molecules exposed on the outer surface of the OM, polymyxins damage thestructure and function of the OM and, as a result, permeabilize (i.e.make permeable) the OM to polymyxin itself, as well as to many othernoxious agents (Nikaido and Vaara 1985, Vaara 1992, Nikaido 2003). Thefinal and lethal target (the bactericidal target) of polymyxins isbelieved to be the cytoplasmic membrane (the inner membrane) ofbacteria.

Numerous efforts have been made to reduce the toxicity of polymyxins.The treatment of polymyxin E (colistin) with formaldehyde and sodiumbisulfite yields colistin sulphomethate, in which the free amino groupsof the five diaminobutyric acid residues have partially been substitutedby sulphomethyl groups (Table 1). The preparations consist of undefinedmixtures of the mono-, di-, tri-, tetra-, and penta-substitutedcompounds. The sulphomethylated preparations, when freshly dissolved inwater, initially lack both the antibacterial activity and toxicity ofthe parent molecule, but when the compounds start decomposing in thesolution, in the blood or in the tissues to yield less substitutedderivatives and free colistin, both the antibacterial activity and thetoxicity are partially brought back. Furthermore, the degree of initialsulphomethylation apparently varies between the commercially availablepharmaceutical preparations. Many other ways to block all the free aminogroups have been published. Examples comprise but are not limited to theformation of unstable Shiff bases with amino acids (Storm et al. 1977).

Polymyxin E nonapeptide (PMEN, colistin nonapeptide, Table 1), obtainedby treating polymyxin E enzymatically and lacking the R-FA and R1, wasshown in 1973 to be less toxic than the parent compound in acutetoxicity assay (immediate death presumably due to direct neuromuscularblockade) in mice (Chihara et al. 1973). However, it also lacked theantibacterial activity, as measured as its ability to inhibit bacterialgrowth (Chirara et al. 1973).

Vaara and Vaara, on the other hand, showed, that polymyxin B nonapeptide(PMBN, Table 1) retains the ability to permeabilize the OM ofGram-negative bacteria (Vaara and Vaara 1983a,b,c; U.S. Pat. No.4,510,132; Vaara 1992). Accordingly, even though it lacks the directantibacterial activity (i.e. the ability to inhibit bacterial growth),it is able to sensitize (i.e. make sensitive or, as also termed, makesusceptible) the bacteria to many antibacterial agents such ashydrophobic antibiotics as well as large antibiotics and some othernoxious agents.

PMBN also sensitizes bacteria to the bactericidal activity of the humancomplement system, present in fresh human serum as a first-line defensesystem against invaders (Vaara and Vaara 1983a, Vaara et al. 1984, Vaara1992). Furthermore, it sensitizes the bacteria to the joint bactericidalactivity of serum complement and human polymorphonuclear white cells(Rose et al. 1999).

PMBN resembles PMEN in being less toxic in the acute toxicity assay inmice than unmodified polymyxins. In further toxicological assays,several criteria proved PBMN to be less toxic than its parent compound,but this polymyxin derivative was still judged to be too nephrotoxic forclinical use (Vaara 1992).

PMBN carries five (5) positive charges. Subsequent studies revealed,quite expectedly, that PMEN, also carrying five (5) positive charges aswell as deacylpolymyxin B and deacylpolymyxin E, both carrying six (6)positive charges are potent agents to sensitize bacteria to otherantibiotics (Viljanen et al. 1991, Vaara 1992). In addition, it has beenshown that a structurally further reduced derivative polymyxin Boctapeptide (PMBO) retains a very effective permeabilizing activitywhile polymyxin B heptapeptide (PMBH) is less active (Kimura et al.1992). PMBN, PMEN and PMBO have five (5) positive charges while PMBH hasonly four (4) positive charges. This difference may explain the weakeractivity of PMBH.

The group of Ofek, Tsubery and Friedkin recently describedpolymyxin-like peptides that were linked to chemotactic peptides, suchas fMLF, that attract polymorphonuclear leucocytes (US patentpublication 2004082505, Tsubery et al. 2005). They described peptidesfMLF-PMBN, MLF-PMBN, fMLF-PMEN, fMLF-PMBO and MLF-PMBO, all carryingfour (4) positive charges, that sensitize Gram-negative bacteria toantibiotics, even though no comparative studies with increasingconcentrations of the compounds were published (Tsubery et al. 2005).

In order to study the structures and functional properties ofpolymyxins, a few works have disclosed, among other compounds, polymyxinderivatives having less than four (4) positive charges.

Teuber (1970) has described the treatment of polymyxin B with aceticanhydride that yields a preparation containing polymyxin B as well asits mono-, di-, tri-, tetra-, and penta-N-acetylated forms. Teuber alsoseparated each group and nonquantitatively reported using an agardiffusion assay that penta-acetylated and tetra-acetylated forms lackedthe ability to halt the growth of Salmonella typhimurium, whereas di-and monoacetylated forms did have such ability. Triacetylated form hadsome ability.

Srinivasa and Ramachandran (1978) isolated partially formylatedpolymyxin B derivatives and showed that a diformyl derivative as well asa triformyl derivative inhibited the growth of Pseudomonas aeruginosa.They did not disclose the compounds' ability to sensitize bacteria toantibiotics. Furthermore, in 1980 they showed that the free amino groupsof triformylpolymyxin B in residues R1 and R3, as well as the free aminogroups of diformylpolymyxin B in residues R1, R3, and R5 are essentialwhile the free amino groups in R8 and R9 are not essential for thegrowth inhibition (Srinivasa and Ramachandran, 1980a).

A shortened polymyxin B derivative octanoyl polymyxin B heptapeptide hasbeen disclosed by Sakura et al. (2004). The attachment of the octanoylresidue to the N-terminus of the residue R4 of the polymyxin Bheptapeptide results in a compound having only three (3) positivecharges. Sakura et al. found that octanoyl polymyxin B heptapeptideinhibits the growth of bacteria only at a very high concentration (128μg/ml), whereas the other derivatives such as octanoyl polymyxin Boctapeptide and octanoyl polymyxin B nonapeptide, both having fourcharges (4) were very potent agents to inhibit bacterial growth.

US patent publication 2006004185 recently disclosed certain polymyxinderivatives and intermediates that can be used to synthesize new peptideantibiotics. The antibacterial compounds described possessed four (4) orfive (5) positive charges.

There is still an urgent need for effective treatments for bacterialinfections, in particular for the infections caused by multiresistantGram-negative bacteria.

SUMMARY

The present invention relates to a polymyxin derivative wherein thetotal number of positive charges at physiological pH is at least two butno more than three, with the proviso that R8 and R9 are not bothformylated when R(FA)-R1-R2-R3 constitutes the native polymyxin B sidechain; and R4 is not directly linked to octanoyl residue when R4-R10constitues a native polymyxin B ring structure. More specifically, thepresent invention relates to a derivative, wherein R1-R10 is selectedfrom the group consisting of SEQ ID NO:s 9-26, preferably SEQ ID NO:s9-20.

The invention also relates to a combination product comprising two ormore of the derivatives according to the present invention, and to apharmaceutical composition comprising such derivative(s) or acombination thereof and pharmaceutically acceptable carriers andexcipients.

Furthermore, the present invention relates to a method for treating,alleviating or ameliorating an infection in an individual caused by aGram-negative bacterium, comprising administering a therapeuticallyeffective amount of a derivative or a combination according to thepresent invention to said individual, wherein said bacterium may beselected from the group consisting of: Escherichia coli, Klebsiellapneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacterfreundii, Pseudomonas aeruginosa, and Acinetobacter baumannii.

In another embodiment, the present invention relates to a method forsensitizing Gram-negative bacteria to an antibacterial agent, comprisingadministering, simultaneously or sequentially in any order, atherapeutically effective amount of said antibacterial agent and aderivative according to the present invention, wherein saidantibacterial agent may be selected from the group consisting ofclarithromycin, azithromycin, erythromycin and other macrolides,ketolides, clindamycin and other lincosamines, streptogramins, rifampin,rifabutin, rifalazile and other rifamycins, fusidic acid, mupirocin,oxazolidinones, vancomycin, dalbavancin, telavancin, oritavancin andother glycopeptide antibiotics, fluoroquinolones, bacitracin,tetracycline derivatives, betalactam antibiotics, novobiocin,pleuromutilins, folate synthesis inhibitors, deformylase inhibitors, andbacterial efflux pump inhibitors.

Also provided are methods for developing novel antibiotics; for reducingthe toxicity of natural polymyxins, octapeptins and their derivatives;for improving the pharmacokinetic properties of natural polymyxins,octapeptins and their derivatives; and for sensitizing clinicallyimportant Gram-negative bacteria to a host defense mechanism complementpresent in the serum.

The present invention also provides uses of a polymyxin derivativeaccording to the present invention in the manufacture of medicament fortreating infections caused by Gram-negative bacteria, such e.g.,Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca,Enterobacter cloacae, Citrobacter freundii, Pseudomonas auruginosa andAcinetobacter baumannii; for the manufacture of a medicament forsensitizing Gram-negative bacteria against anti-bacterial agents; andfor sensitizing Gram-negative bacteria to a host defense mechanismcomplement present in the serum.

Finally, the present invention relates to a process for preparing apolymyxin derivative according to the present invention, comprisingmodifying a natural or synthetic polymyxin or octapeptin compound or aderivative thereof having 4 to 6 positively charged residues byreplacing 1 to 4 of said residues by neutral residues or a covalentbond, or by converting 1 to 4 of said residues into neutral residues inorder to obtain a polymyxin derivative of formula (I) having 2 or 3positively charged residues.

DEFINITIONS

“Physiological pH” as used herein refers to a pH value of more than 7,0and below 7,6, such as a pH value in the range of from 7.1 to 7.5, forexample in the range of from 7.2 to 7.4.

“Positive charge” as used herein denote positive charges at theabove-defined physiological pH.

“Cationic” molecule as used herein refers to a molecule that containsone or more positive charges.

“Amino acid residue” as used herein refers to any natural, non-naturalor modified amino acid residue, either in L- or D-configuration.

“Equivalent residues” as used herein, is intended to include obviousmodifications to e.g., amino acids, resulting in non-natural amino acidsor derivatives thereof, but retaining the structural and/or functionalcapacity of the replaced residue.

“Natural polymyxin(s)” as used herein, refers to polymyxins andcirculins.

“Polymyxin derivative” refers, for the purpose of this invention, tosynthetic or semisynthetic derivatives of natural polymyxins oroctapeptins, which have a cyclic heptapeptide (or heptapeptide ring)portion R4—R10 and a side chain linked to the N-terminal aminoacylresidue R4. The side chain may consist of an R(FA)-triaminoacyl(R1—R3),an R(FA)-diaminoacyl(R2—R3), an R(FA)-monoamino-acyl(R3), or of R(FA)alone.

“Compounds” as used herein include all stereochemical isomers of saidcompound.

“Sensitizing activity” or “ability to sensitize” as used herein isintended to include any ability to increase the sensitivity, makesensitive or make susceptible a bacterium to an antibacterial agent.

Abbreviations

Fatty acids: FA, fatty acyl residue; 6-MOA and MOA, 6-methyloctanoylresidue; 6-MHA and MHA, 6-methylheptanoyl residue; MO(H)A, the mixtureof 6-methyloctanoyl, 6-methylheptanoyl and related fatty acyl residuesoccurring in polymyxin B; OHMDA, 3-OH-8-methyldecanoic acid; OA,octanoyl residue; DA, decanoyl residue;

Amino acids: Dab, α,γ-diamino-n-butyryl residue; fDab,N-γ-formyldiamino-n-butyryl residue; acDab, N-γ-acetyldiamino-n-butyrylresidue; Abu, α-aminobutyryl residue; Thr, threonyl residue; Ser,serinyl residue; Phe, phenylalanyl residue; Leu, leucyl residue; Ile,isoleucyl residue; Ala, alanyl residue; smDab, γ-sulphomethylatedα,γ-diamino-n-butyryl residue. One-letter codes for modified amino acylresidues: X, Dab; Z, Abu; B, N-γ-f Dab; J, N-γ-acDab.

Peptides: DAPB, deacylpolymyxin B; DAC, deacylcolistin; PMBN, polymyxinB nonapeptide; PMEN, polymyxin E nonapeptide; PMBO, polymyxin Boctapeptide; PMHP, polymyxin B heptapeptide.

Other: cy, cyclo (to denote the cyclic part of the peptide, enclosedwithin brackets); f, formyl; ac, acetyl; LPS, lipopolysaccharide; OM,outer membrane; CFU, colony forming unit. The symbol * is used herein tomark the residues between which the heptapeptide ring portion of thecompound is closed leaving the remaining part of the molecule as a sidechain.

DETAILED DESCRIPTION OF THE INVENTION

It has now been found that polymyxin derivatives containing at least two(2) but no more than three (3) positive charges still possessantibacterial activity against Gram-negative bacteria, and/or possessthe ability to sensitize Gram-negative bacteria to antibacterial agents,such as antibiotics, semisynthetic antibiotics, chemotherapeutic agentsand host defense factors, such as complement.

This reduction of positive charges may improve the pharmacologicalproperties of the derivatives according to the present invention whencompared to natural polymyxins and their known derivatives. Morespecifically, it may reduce the toxicity, including nephrotoxicity, ofthe compounds, and/or reduce the histamine liberation from the hosttissue, exerted by the compounds, and/or result in more suitablepharmacokinetic properties such as longer serum half life or lowersusceptibility to the inactivation by polyanionic tissue and pusconstituens, as compared to clinically used polymyxins and theirpreviously described and characterized derivatives, such as polymyxin Bnonapeptide.

The present invention thus relates to a polymyxin derivative which maybe represented by the general formula I:

wherein R1, R2 and R3 may be absent; and

wherein the total number of free, unsubstituted cationic charges in thecompound is at least two (2) and does not exceed three (3); or apharmaceutically acceptable salt thereof.

In natural polymyxins and octapeptins, R(FA) is 6-methyloctanoic acid(6-MOA), 6-methylheptanoic acid (6-MHA), octanoic acid, heptanoic acid,nonanoic acid, 3-OH-6-methyloctanoic acid, 3-OH-8-methyldecanoic acid,3-OH-8-methylnonanoic acid, 3-OH-8-decanoic acid, and3-OH-6-methyloctanoic acid. Examples of known derivatives that haveantibacterial activity include those wherein R(FA) is γ-phenylbutyricacid, isovaleric acid, 9-fluorenyl-methoxycarbonic acid, a series of C:9to C:14 unbranched fatty acids as well as iso C:9 and iso C:10 fattyacids.

In a derivative according to the present invention, R(FA) may be anyhydrophobic fatty acid residue, and is preferably selected from thegroup consisting of octanoyl, decanoyl and 6-MHA residues.

A person skilled in the art may readily recognize equivalents of thesepreferred hydrophobic R(FA) residues, which may be selected from thegroup consisting of e.g. an optionally substituted acyl or alkylresidue, an optionally substituted isoalkyl residue, an optionallysubstituted cycloalkyl residue, an optionally substituted alkenylresidue, an optionally substituted cycloalkenyl residue, an optionallysubstituted aryl residue, an optionally substituted heteroaryl residue,an optionally substituted heterocyclic residue, wherein said residuespreferably have more than five (5) carbon atoms and wherein thesubstitutions may also include those optionally designed between theresidue and the N-terminus of the peptide. R(FA) may also be a stretchof a hydrophobic oligopeptide. Examples of possible R(FA) residuesinclude (but are not limited to) octanoyl, nonanoyl, isononanoyl,decanoyl, isodecanoyl, undecanoyl, dodecanoyl, tetradecanoyl,cyclohexanoyl, cycloheptanoyl, cyclooctanoyl, cyclononanoyl,cycloisononanoyl, cyclodecanoyl, cycloisodecanoyl, cycloundecanoyl,cyclododecanoyl, cyclotetradecanoyl, hexanoyl, heptanoyl, and9-fluorenylmethoxycarbonyl residues.

In natural polymyxins and octapeptins, R1 is Dab or absent (i.e.replaced by a covalent bond). Examples of known derivatives that haveantibacterial activity include those wherein R1 is Ala or a covalentbond.

In a derivative according to the present invention R1, if present, maybe any amino acid residue, provided that the total number of positivecharges in said derivative does not exceed three and that the totalnumber of positive charges in the side chain portion does not exceedtwo, and is preferably Abu, if present.

In natural polymyxins and octapeptins, R2 is Thr or absent (i.e.replaced by a covalent bond). Examples of known derivatives that haveantibacterial activity include those wherein R2 is 0-acetyl-Thr,0-propionyl-Thr, O-butyryl-Thr or a covalent bond.

In a derivative according to the present invention, R2, if present, maybe any amino acid residue, provided that the total number of positivecharges in said derivative does not exceed three and that the totalnumber of positive charges in the side chain portion does not exceedtwo, and is preferably selected from the group consisting of Thr, DThr,and DAIa, if present. A person skilled in the art may also recognize anequivalent residue of Thr to be Ser.

In natural polymyxins and octapeptins, R3 is Dab, DDab or DSer. Examplesof numerous known synthetic derivatives that have antibacterial activityinclude those wherein R3 is Lys or 2-amino-4-guanidino butyric acid.

In a derivative according to the present invention, R3, if present, maybe any amino acid residue, provided that the total number of positivecharges in said derivative does not exceed three and that the totalnumber of positive charges in the chain portion does not exceed two, andis preferably selected from the group consisting of Thr, DThr, Ser,DSer, DAIa, Dab and Abu, if present.

A person skilled in the art may readily recognize equivalent residues ofthese preferred residues R1, R2 and R3, and may select such from a groupconsisting of e.g. a covalent bond, alanine, 2-aminoadipic acid,α-n-butyric acid, N-(4-aminobutyl)glycine, α-aminobutyric acid,γ-aminobutyric acid, α-aminocaproic acid, aminocyclopropanecarboxylate,aminoisobutyric acid, aminonorbornylcarboxylate, α-amino-n-valeric acid,arginine, N_(ω)-methylarginine, asparagine, a-methylaspartate, asparticacid, N-benzylglycine, N-(2-carbamylethyl)glycine,N-(carbamylethyl)glycine, 1-carboxy-1(2,2-diphenylethylamino)cyclopropane, cysteine, N_(α)-methyldiamino-n-butyric acid,N_(γ)-acetyldiamino-n-butyric acid, N_(γ)-formyldiamino-n-butyric acid,N_(γ)-methyldiamino-n-butyric acid,N-(N-2,2-diphenylethyl)carbamylmethyl-glycine, N-(N-3,3-diphenylpropyl)carbamylmethyl(1)glycine, N-(3,3-diphenylpropyl) glycine, glutamic acid,glutamine, glycine, t-butylglycine, 2-amino-4-guanidinobutyric acid,N-(3-guanidinopropyl)glycine, histidine, homophenylalanine,isodesmosine, isoleucine, leucine, norleucine, hydroxylysine,N_(α)-methyllysine, lysine, N_(α)-methylhydroxylysine,N_(α)-methyllysine, N_(ε)-acetylhydroxylysine, N_(ε)-acetyllysine,N_(ε)-formylhydroxylysine, N_(ε)-formyllysine,N_(ε)-methylhydroxylysine, N_(ε)-methyllysine, methionine,α-methyl-γ-aminobutyrate, α-methyl-aminoisobutyrate,α-methylcyclohexylalanine, α-napthylalanine, norleucine, norvaline,α-methylornithine, N_(α)-methylornithine, N_(δ)-acetylornithine,N_(δ)-formyl-ornithine, N_(δ)-methylornithine, ornithine, penicilamine,phenylalanine, hydroxyproline, proline, N_(α)-methyldiamino-n-propionicacid, N_(β)-acetyldiamino-n-propionic acid,N_(β)-formyldiamino-n-propionic acid, N_(β)-methyldiamino-n-propionicacid, phosphoserine, serine, phosphothreonine, threonine, tryptophan,tyrosine, norvaline, and valine.

In natural polymyxins and octapeptins, R4 is Dab. Examples of syntheticderivatives that have antibacterial activity include those wherein R4 isLys.

In a derivative according to the present invention R4 is an amino acidresidue comprising a functional side chain able to cyclicize themolecule, and may be selected from the group of equivalent residuesconsisting of Lys, hydroxylysine, ornithine, Glu, Asp, Dab,diaminopropionic acid, Thr, Ser and Cys, preferably Dab.

In natural polymyxins and octapeptins, R5, R8 and R9 are Dab. Examplesof synthetic derivatives that have antibacterial activity include thosewherein R5, R8, and R9 may be Lys or 2-amino-4-guanidino butyric acid.

In a derivative according to the present invention R5, R8 and R9 may bea positively charged or a neutral amino acid residue, preferably Dab orAbu, provided that the total number of positive charges in saidderivative does not exceed three.

A person skilled in the art, may readily recognize equivalent residuesof these preferred residues, and may select such from a group consistingof e.g. diaminobutyric acid, diaminopropionic acid, lysine,hydroxylysine, ornithine, 2-amino-4-guanidinobutyric acid, glycine,alanine, valine, leucine, isoleucine, phenylalanine, D-phenylalanine,methionine, threonine, serine, α-amino-n-butyric acid, α-amino-n-valericacid, α-amino-caproic acid, N_(ε)-formyl-lysine, N_(ε)-acetyllysine,N_(ε)-methyllysine, N_(ε)-formylhydroxylysine,N_(ε)-acetylhydroxylysine, N_(ε)-methylhydroxylysine,L-N_(α)-methylhydroxylysine, N_(γ)-formyldiamino-n-butyric acid,N_(γ)-acetyldiamino-n-butyric acid, N_(γ)-methyldiamino-n-butyric acid,N_(β)-formyldiamino-n-propionic acid, D-N_(β)formyldiamino-n-propionicacid, N_(β)-acetyldiamino-n-propionic acid,N_(β)methyldiamino-n-propionic acid, N_(δ)-formylornithine,N_(δ)-acetylornithine and N_(δ)-methylornithine.

In natural polymyxins and octapeptins, R6 is DPhe or DLeu and R7 is Leu,Ile, Phe or Thr. Synthetic derivatives that have antibacterial activityinclude those wherein R6 is DTrp and wherein R7 is Ala.

In a derivative according to the present invention, R6 is an optionallysubstituted hydrophobic amino acid residue, preferably DPhe or DLeu, andR7 is an optionally substituted hydrophobic residue, preferably Leu, Thror Ile.

A person skilled in the art may readily recognize equivalent residues ofthese preferred hydrophobic residues, and may select such from a groupconsisting of e.g. phenylalanine, α-amino-n-butyric acid, tryptophane,leucine, methionine, valine, norvaline, norleucine, isoleucine andtyrosine. A person skilled in the art may also recognize the equivalentresidue of threonine to be serine.

In natural polymyxins and octapeptins, R10 is Thr and Leu. Examples ofknown derivatives that have antibacterial activity include those whereinR10 is 0-acetyl-Thr, 0-propionyl-Thr or 0-butyryl-Thr.

In a derivative according to the present invention, R10 is Leu or anynon-hydrophobic amino acid residue, provided that that the total numberof positive charges in said derivative does not exceed three. PreferablyR10 is Thr or Leu.

A person skilled in the art may also recognize the equivalent residue ofthreonine to be serine.

More specifically, preferred residues are chosen in such a manner thatR8 and R9 are not both formylated when R(FA)-R1—R2—R3 constitutes thenative polymyxin B sidechain; and R4 is not directly linked to octanoylresidue when R4—R10 constitues a native polymyxin B ring structure.

The specific positions of the at the most three (3) positive chargesreferred to herein above can be located in the heptapeptide ring portionand/or in the side chain, if present. When three (3) positive chargesare present in the derivatives according to the invention, said three(3) positive charges can be located in the heptapeptide ring portion; ortwo (2) positive charges can be located in heptapeptide ring portionwhile the remaining one positive charge is located in the side chain; orone (1) positive charge can be located in the heptapeptide ring portionwhile the remaining two (2) positive charges are located in the sidechain. Preferably at least two (2) positive charges are located in theheptapeptide ring portion.

In one embodiment, derivatives according to the present invention can beselected from the group of derivatives wherein R1—R10 is selected fromthe group consisting of Thr-DSer-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e.SEQ ID NO. 10; Thr-DThr-cy[Dab-Dab-DPhe-Thr-Dab-Dab-Thr-], i.e. SEQ IDNO. 11; Thr-DSer-cy[Dab-Dab-DPhe-Thr-Dab-Dab-Thr-], i.e. SEQ ID NO. 12;Thr-Abu-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. SEQ ID NO. 13;Abu-Thr-Abu-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. SEQ ID NO. 14;Thr-Dab-cy[Dab-Dab-DPhe-Leu-Abu-Dab-Thr-], i.e. SEQ ID NO. 15;Thr-Abu-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Leu-], i.e. SEQ ID NO. 16;Thr-DAla-cy[Dab-Dab-DPhe-Thr-Dab-Dab-Thr-], i.e. SEQ ID NO. 17;Thr-Dab-cy[Dab-Dab-DPhe-Leu-Dab-Abu-Thr-], i.e. SEQ ID NO. 18;Thr-Abu-cy[Dab-Dab-DLeu-Leu-Dab-Dab-Thr-], i.e. SEQ ID NO. 19;DAla-DAla-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. SEQ ID NO. 20;cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. SEQ ID NO. 9;Abu-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. SEQ ID NO. 21;Thr-Dab-cy[Dab-Abu-DPhe-Leu-Dab-Dab-Thr-], i.e. SEQ ID NO. 22;Dab-Thr-Dab-cy[Dab-Dab-DPhe-Leu-Abu-Abu-Thr-], i.e. SEQ ID NO. 23;Thr-Abu-cy[Dab-Lys-DPhe-Leu-Dab-Dab-Thr-], i.e. SEQ ID NO. 24;Thr-Abu-cy[Dab-Abu-DPhe-Leu-Dab-Dab-Thr-], i.e. SEQ ID NO. 25; andThr-Abu-cy[Dab-Dab-DPhe-Leu-Dab-Abu-Thr-], i.e. SEQ ID NO. 26.

In other embodiments, derivatives according to the present invention canbe selected from the group consisting of:OA-Thr-DSer-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. OA-SEQ ID NO. 10;DA-Thr-DSer-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. DA-SEQ ID NO. 10;OA-Thr-DThr-cy[Dab-Dab-DPhe-Thr-Dab-Dab-Thr-], i.e. OA-SEQ ID NO. 11;OA-Thr-DSer-cy[Dab-Dab-DPhe-Thr-Dab-Dab-Thr-], i.e. OA-SEQ ID NO. 12;DA-Thr-Abu-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. DA-SEQ ID NO. 13;OA-Thr-Abu-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. OA-SEQ ID NO. 13;MHA-Thr-Abu-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. MHA-SEQ ID NO. 13;MHA-Abu-Thr-Abu-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. MHA-SEQ ID NO.14; OA-Thr-Dab-cy[Dab-Dab-DPhe-Leu-Abu-Dab-Thr-], i.e. OA-SEQ ID NO. 15;OA-Thr-Abu-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Leu-], i.e. OA-SEQ ID NO. 16;OA-Thr-DAla-cy[Dab-Dab-DPhe-Thr-Dab-Dab-Thr-], i.e. OA-SEQ ID NO. 17;OA-Thr-Dab-cy[Dab-Dab-DPhe-Leu-Dab-Abu-Thr-], i.e. OA-SEQ ID NO. 18;OA-Thr-Abu-cy[Dab-Dab-DLeu-Leu-Dab-Dab-Thr-], i.e. OA-SEQ ID NO. 19;OA-DAla-DAla-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. OA-SEQ ID NO. 20;DA-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. DA-SEQ ID NO. 9;OA-Abu-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. OA-SEQ ID NO. 21;OA-Thr-Dab-cy[Dab-Abu-DPhe-Leu-Dab-Dab-Thr-], i.e. OA-SEQ ID NO. 22;MHA-Dab-Thr-Dab-cy[Dab-Dab-DPhe-Leu-Abu-Abu-Thr-], i.e. MHA-SEQ ID NO.23; OA-Thr-Abu-cy[Dab-Lys-DPhe-Leu-Dab-Dab-Thr-], i.e. OA-SEQ ID NO. 24;OA-Thr-Abu-cy[Dab-Abu-DPhe-Leu-Dab-Dab-Thr-], i.e. OA-SEQ ID NO. 25; andOA-Thr-Abu-cy[Dab-Dab-DPhe-Leu-Dab-Abu-Thr-], i.e. OA-SEQ ID NO. 26.

Preferably, derivatives according to the present invention are selectedfrom the group consisting of:OA-Thr-DSer-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. OA-SEQ ID NO. 10;DA-Thr-DSer-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. DA-SEQ ID NO. 10;OA-Thr-DThr-cy[Dab-Dab-DPhe-Thr-Dab-Dab-Thr-], i.e. OA-SEQ ID NO. 11;OA-Thr-DSer-cy[Dab-Dab-DPhe-Thr-Dab-Dab-Thr-], i.e. OA-SEQ ID NO. 12;DA-Thr-Abu-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. DA-SEQ ID NO. 13;OA-Thr-Abu-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. OA-SEQ ID NO. 13;MHA-Thr-Abu-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. MHA-SEQ ID NO. 13;MHA-Abu-Thr-Abu-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. MHA-SEQ ID NO.14; OA-Thr-Dab-cy[Dab-Dab-DPhe-Leu-Abu-Dab-Thr-], i.e. OA-SEQ ID NO. 15;OA-Thr-Abu-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Leu-], i.e. OA-SEQ ID NO. 16;OA-Thr-DAla-cy[Dab-Dab-DPhe-Thr-Dab-Dab-Thr-], i.e. OA-SEQ ID NO. 17;OA-Thr-Dab-cy[Dab-Dab-DPhe-Leu-Dab-Abu-Thr-], i.e. OA-SEQ ID NO. 18;OA-Thr-Abu-cy[Dab-Dab-DLeu-Leu-Dab-Dab-Thr-], i.e. OA-SEQ ID NO. 19;OA-DAla-DAla-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. OA-SEQ ID NO. 20;and DA-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. DA-SEQ ID NO. 9.

As shown in the example section herein, the compounds according to thepresent invention carrying only three (3) positive charges can be verypotent agents to inhibit the growth of Gram-negative bacteria or tosensitize them to antibacterial agents, and even derivatives carryingonly two (2) positive charges can have the same effect, although at amore moderate level.

For direct antibacterial activity at least two (2) and more preferablythree (3) positive charges are located in the heptapeptide ring portion,and for sensitizing activity at least one (1) and more preferably two(2) or three (3) positive charges are located in the heptapeptide ringpart. Furthermore, the presence of two hydroxyl groups in the side chainpart significantly enhances the direct antibacterial activity.

The works of Teuber (1970), Srinivasa and Ramachandran (1980a), andSakura et al. (2004) disclose, among other polymyxin derivatives,derivatives having only two (2) or three (3) positive charges. However,the antibacterial activity of the disclosed derivatives is very weak andclinically irrelevant (Sakura et al. 1980), attributed to amino acidresidues in full contradiction to the findings of the present invention(Srinivasa and Ramachandran 1980a) or attributed to no specific aminoacid residues at all, due to incomplete purification (Teuber 1970).Furthermore, none of the works cited above describe, suggest or motivateto study the ability of such derivatives to sensitize Gram-negativebacteria to antibacterial agents. As the examples of the presentinvention clearly show, one cannot predict the ability of a polymyxinderivative to sensitize Gram-negative bacteria to antibacterial agentson the basis of its ability to inhibit the growth of the same. Forexample, whereas a clinically irrelevant concentration of octanoylpolymyxin B heptapeptide of as high as 128 μg/ml is needed to inhibitthe growth of E. coli (Sakura et al. 2004), an amount as low as 4 μg isenough for a moderate sensitization of the bacterium to rifampin, asshown in the example section herein.

The acetylated polymyxin derivatives described by Teuber (1970) aremixtures of differently acetylated derivatives. Acetic anhydride canreact with any of the five free amino groups of the polymyxin B moleculeto yield a monoacetylated polymyxin. Therefore a monoacetylatedpolymyxin according to Teuber is a mixture of five monoacetylatedpolymyxin derivatives, each acetylated at a different amino group. Adiacetylated polymyxin according to Teuber is a mixture of tendifferently diacetylated derivatives and triacetylated polymyxin is alsoa mixture of ten differently triacetylated derivatives. Teuber did notattempt to isolate these derivatives from the mixtures. The problem withsuch modified polymyxins is that the partial modification may result inreduced specificity. Therefore, some of the amino groups that areimportant for the anti-bacterial activity are partially substituted (andhence inactivated) whereas some of the non-important amino groups remainpartially unsubstituted. Furthermore, the degree of substitution maylead to lot-to-lot variations.

The polymyxin derivatives according to the present invention, on theother hand, are isolated and structurally clearly defined and identifiedcompounds.

Srinivasa and Ramachandran (1980a) proposed that the free side chainamino groups of triformyl-polymyxin B in residues R1 and R3, as well asthe free amino groups of diformylpolymyxin B in residues R1, R3, and R5are essential while the free amino groups in R8 and R9 are not essentialfor the growth inhibition of Pseudomonas aeruginosa. As a contrast totheir conclusions, the compounds in the present invention include thosethat lack the free amino groups in R1 and R3, carry them in R5, R8 andR9, and yet are potent agents against Ps. aeruginosa as well as otherGram-negative bacteria.

A shortened polymyxin B derivative octanoyl polymyxin B heptapeptide hasbeen disclosed by Sakura et al. (2004). The attachment of the octanoylresidue to the N-terminus of the residue R4 of the polymyxin Bheptapeptide results in a compound having only three (3) positivecharges. Sakura et al. found that octanoyl polymyxin B heptapeptideinhibits the growth of bacteria only at a very high (and clinicallyirrelevant) concentration (128 μg/ml), whereas the other derivativessuch as octanoyl polymyxin B octapeptide and octanoyl polymyxin Bnonapeptide, both having four charges (4) are very potent agents toinhibit bacterial growth. Sakura et al. did not disclose or suggest theability of octanoyl polymyxin B heptapeptide to sensitize bacteria toantibacterial agents, nor teach that a longer fatty acid tail increasesits antibacterial activity, as shown in the example section herein.

The present invention in one aspect provides new polymyxin derivativeshaving two (2) or three (3) positive charges only and still beingcapable of inhibiting the growth of one or more Gram-negative bacterialspecies and or sensitizing one or more Gram-negative bacterial speciesto an antibiotic or antibacterial agent.

The susceptibility of bacteria to an antibacterial agent may bedetermined by two microbiological methods. A rapid but crude procedureuses commercially available filter paper disks that have beenimpregnated with a specific quantity of the antibacterial agent. Thesedisks are placed on the surface of agar plates that have been inoculatedwith a suspension of the organism being tested, and the plates areobserved for zones of growth inhibition. A more accurate technique, thebroth dilution susceptibility test, involves preparing test tubescontaining serial dilutions of the drug in liquid culture media, theninoculating the organism being tested into the tubes. The lowestconcentration of drug that inhibits growth of the bacteria after asuitable period of incubation is reported as the minimum inhibitoryconcentration (MIC).

Derivatives according to the present invention may inhibit the growth ofor sensitize to antibacterial agents clinically important Gram-negativebacteria such as those belonging to the genus of Acinetobacter,Aeromonas, Alcaligenes, Bordetella, Branhamella, Campylobacter,Citrobacter, Enterobacter, Escherichia, Francisella, Fusobacterium,Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella,Pasteurella, Plesiomonas, Pseudomonas, Salmonella, Serratia, Shigella,and Yersinia species. The bacteria may be, for example, Escherichiacoli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae,Enterobacter aerogenes, other species of Enterobacter, Citrobacterfreundii, Pseudomonas aeruginosa, other species of Pseudomonas,Acinetobacter baumannii, as well as many other species ofnon-fermentative Gram-negative bacteria. The bacteria also includeHelicobacter pylori, as well as other clinically important Gram-negativebacteria.

The bacterial infections to be treated include, for example, bacteremia,septicemia, skin and soft tissue infection, pneumonia, meningitis,infections in the pelveoperitoneal region, foreing body infection, feverin hematological patient, infection associated with an intravenous lineor other catheter, canyl and/or device, infection in gastrointestinaltract, in the eye, or in the ear, superficial skin infection, andcolonization of gastrointestinal tract, mucous membranes and/or skin bypotentially noxious bacteria.

The bacterial infectious diseases include (but are not limited to)severe hospital-acquired infections, infections of the immunocompromisedpatients, infections of the organ transplant patients, infections at theintensive care units (ICU), severe infections of burn wounds, severecommunity-acquired infections, infections of cystic fibrosis patients,as well as infections caused by multi-resistant Gram-negative bacteria.

The present invention is also directed to combinations of two or morederivatives according to the present invention for combinationtreatment. The combinations may include derivatives having differentspectra of antibacterial activity or a capability to sensitize differentspecies or strains of Gram-negative bacteria to antibacterial agents.

Another aspect of the present invention is directed to pharmaceuticalcompositions comprising polymyxin derivatives according to the presentinvention, their salt forms, selected combinations thereof, andoptionally an antibacterial agent formulated together with one or morepharmaceutically acceptable carriers and excipients. They facilitateprocessing of the active compounds into preparations which can be usedpharmaceutically and include e.g. diluting, filling, buffering,thickening, wetting, dispersing, solubilizing, suspending, emulsifying,binding, stabilizing, disintegrating, encapsulating, coating, embedding,lubricating, colouring, and flavouring agents as well as absorbents,absorption enhancers, humefactants, preservatives and the like,well-known to a person skilled in the art.

Pharmaceutical compositions include compositions wherein the activeingredients are contained in an amount effective to achieve the intendedpurpose. More specifically, a therapeutically effective amount means anamount of compound effective to treat, prevent, alleviate or amelioratesymptoms of pathology or prolong the survival of the subject beingtreated at a reasonable benefit to risk ratio applicable to any medicaltreatment. Determination of a therapeutically effective amount is wellwithin the capability of those skilled in the art of medicine.

Compositions may be produced by processes well known in the art, e.g. bymeans of conventional mixing, dissolving, encapsulating, entrapping,lyophilizing, emulsifying and granulating processes. The properformulation is dependent upon the route of administration chosen, andthe pharmaceutical composition can be formulated for immediate releaseor slow release (e.g. in order to prolong the therapeutic effect and/orimprove tolerability). Furthermore, the formulations may conveniently bepresented in unit dosage form by methods known in the art of pharmacy.

Pharmaceutical compositions according to the present invention include(but are not limited to) those intended for intravenous, intramuscular,oral, or topical administration as well as those being administered as asuppositorium or as an inhalable aerosol. The compositions includeintravenous, intramuscular, intraperitoneal, subcutaneous,intramedullary, intrathecal, intraventricular, intranasal, orintraocular injections, inhalable aerosols as well as those intended forrectal, oral, intravaginal, transmucosal or transdermal delivery.

For parenteral administration (e.g. by bolus injection, fast runninginfusions, or slow infusions), the compounds according to this inventionas well as the combinations described above may be formulated as theirsuitable salt or ester forms in sterile aqueous solutions, preferablyphysiologically compatible fluids such as saline, 5% dextrose, Ringer'ssolution, and Hank's solution. The formulation may also include organicsolvents such as propylene glycol, polyethylene glycol, propylene glycolor related compounds as well as preservatives and surfactants.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

In addition, the pharmaceutical compositions for parental administrationmay be suspensions or emulsions in oily or aqueous vehicles, and maycontain formulatory agents such as suspending, stabilizing and/ordispersing agents. Suitable lipophilic vehicles and solvents includefatty oils such as natural and/or synthetic fatty acids esters, such asethyl oleate and triglycerides, or liposomes. The suspensions maycontain substances, which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol or dextran.

The parenteral compositions can be presented in unit-dose or multidosesealed containers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use.

For oral administration, solid form preparations include e.g. powders,tablets, pills, dragees, lozenges, capsules, cachets, and microgranularpreparations. Pharmaceutical preparations can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. A solid carrier/excipient can be one ormore substances which may also act as diluents, solubilizers,lubricants, suspending agents, binders, preservatives, flavouringagents, wetting agents, tablet disintegrating agents, or anencapsulating material. Suitable carriers include, but are not limitedto, magnesium carbonate, magnesium stearate, talc, dextrose, lactose,pectin, starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.

Liquid preparations suitable for oral administration include e.g.aqueous solutions, syrups, elixirs, aqueous suspensions, emulsions andgels. Aqueous solutions can be prepared by dissolving the activecomponent in water and adding suitable stabilizing and thickening agentsas well as colorants and flavours. Aqueous suspensions can be preparedby dispersing the finely divided active component in water with viscousmaterial, such as natural or synthetic gums, resins, methylcellulose,sodium carboxymethylcellulose, and other well known suspending agents.Emulsions may be prepared in solutions in aqueous propylene glycolsolutions or may contain emulsifying agents such as lecithin, sorbitanmonooleate or acacia.

The compounds according to the invention or combinations described abovemay also be formulated for topical administration. The active compoundsare admixed under sterile conditions with pharmaceutically acceptablecarriers/excipients, including any needed buffering agents andpreservatives. Ointments, creams and lotions may, for example, beformulated with an aqueous or oily base with the addition of suitableemulsifying, dispersing, suspending, thickening, stabilizing, orcoloring agents. Commonly used excipients include animal and vegetablefats and oils, waxes, paraffins, starch, cellulose derivatives,tragacanth, and polyethylene glycol.

Other topical formulations include, but are not limited to, ear-drops,eye-drops and transdermal patches.

For transdermal as well as transmucosal administration, penetrantsgenerally known in the art may be used in the formulation.

For administration by inhalation, the compounds according to thisinvention and the combinations described above are delivered in the formof an aerosol spray presentation from a ventilator, pressurized pack ora nebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds according to this invention and the combinations describedabove may also be formulated in rectal compositions such as retentionenemas or suppositories, using conventional suppository bases such ascocoa butter, other glycerides, polyethylene glycol, or a suppositorywax.

The present invention also relates to a method for using the presentpolymyxin derivatives or a combination of such derivatives as a part ofthe clinical treatment of (or a preventive prophylactic regimen for)human or animal subjects suffering of an infectious disease, andcomprises administering to said subject an therapeutically effectivedose of at least one derivative according to the present invention,optionally in combination with an antibacterial agent.

The present invention also relates to a method of sensitizingGram-negative bacteria to an antibacterial agent, wherein the derivativeaccording to the present invention is administered simultaneously, orsequentially in any order, with a therapeutically effective amount ofsaid antibacterial agent.

The derivative of the present invention and the antibacterial agent maybe administered together as one formulation or by different routes. Forexample, the polymyxin derivative may be administered intravenouslywhile the antibacterial agent is administered intramuscularly,intravenously, subcutaneously, orally or intraperitoneally.Alternatively, the derivative may be administered intramuscularly orintraperitoneally while the antibacterial agent is administeredintravenously, intramuscularly or intraperitoneally, or the derivativemay be administered in an aerosolized or nebulized form while theantibacterial agent is administered, e.g., intravenously. The derivativeand the antibacterial agents may be administered simultaneously orsequentially, as long as they are given in a manner sufficient to allowboth to achieve effective concentrations at the site of infection.

“Therapeutic effectiveness” is based on a successful clinical outcome,and does not require that a derivative according to the presentinvention, optionally in combination with an antibacterial agent, kills100% of the bacteria involved in an infection. Successful treatmentdepends on achieving a level of antibacterial activity at the site ofinfection, sufficient to inhibit the bacteria in a manner that tips thebalance in favor of the host. When host defenses are maximallyeffective, the antibacterial effect required may be modest. Reducingorganism load by even one log (a factor of 10) may permit the host's owndefenses to control the infection. In addition, augmenting an earlybactericidal/bacteriostatic effect can be more important than long-termbactericidal/bacteriostatic effect. These early events are a significantand critical part of therapeutic success, because they allow time forhost defense mechanisms to activate. Increasing the bactericidal ratemay be particularly important for infections such as meningitis, bone orjoint infections.

The therapeutic effectiveness of an antibacterial agent depends on thesusceptibility of the bacterial species to said antibacterial agent atthe clinically relevant concentration of the derivative according tothis invention. The effect of compounds according to the presentinvention to improve the therapeutic effectiveness of antibacterialagents in vivo may be demonstrated in in vivo animal models, such asmouse peritonitis or rabbit bacteremia assays, and may be predicted onthe basis of a variety of in vitro tests, including (1) determinationsof the minimum inhibitory concentration (MIC) of an antibacterial agentrequired to inhibit growth of a Gram-negative bacterium for 24 hours,(2) determinations of the effect of an antibacterial agent on thekinetic growth curve of a Gram-negative bacterium, and (3) checkerboardassays of the MIC of serial dilutions of antibacterial agent alone or incombination with serial dilutions of compound(s). Exemplary models ortests are well known in the art.

Using in vitro determinations of MIC at 24 hours, a derivative accordingto the present invention may be shown to reduce the MIC of theanti-bacterial agent. With this result, it is expected that concurrentadministration of the compound in vivo will increase susceptibility of aGram-negative bacterium to the antibacterial agent. A compound accordingto the present invention may also be shown to reduce the MIC of anantibacterial agent from the range in which the organism is consideredclinically resistant to a range in which the organism is consideredclinically susceptible. With this result, it is expected that concurrentadministration in vivo of the one or more compound(s) according to thepresent invention with the antibacterial agent will reverse resistanceand effectively convert the antibiotic-resistant organism into anantibiotic-susceptible organism.

By measuring the effect of antibacterial agents on the in vitro growthcurves of Gram-negative bacteria, in the presence or absence of acompound according to the present invention, the compound may be shownto enhance the early antibacterial effect of antibacterial agents withina period of preferably less than 24 hours. Enhancement of earlybactericidal/growth inhibitory effects is important in determiningtherapeutic outcome.

A polymyxin derivative according to the present invention and anantibacterial agent may also have synergistic or potentiating effectsbeyond the individual effects of each agent alone or the additiveeffects of the agents together. In a checkerboard assay, the combinationof a compound according to the present invention with antibacterialagents may result in a “synergistic” fractional inhibitory concentrationindex (FIC). The checkerboard method is based on additivity, whichassumes that the result observed with multiple drugs is the sum of theseparate effects of the drugs being tested; according to this system aFIC of less than 0.5 is scored as synergy, 1 is scored as additive, andgreater than 1 but less than 2 is scored as indifferent.

Antibacterial agents suitable for use in combination with derivativesaccording to the present invention, include e.g. macrolides, such asclarithromycin, azithromycin, and erythromycin, ketolides, lincosamines,such as clindamycin, streptogramins, rifamycins, such as rifampin,rifabutin and rifalazile, fusidic acid, mupirocin, oxazolidinones,glycopeptide antibiotics, such as vancomycin, dalbavancin, telavancinand oritavancin, fluoroquinolones, tetracycline derivatives, hydrophobicderivatives of penicillins, cephalosporins, monobactams, carbapenems,penems and other betalactam antibiotics, novobiocin, pleuromutilins,folate synthesis inhibitors, deformylase inhibitors, and bacterialefflux pump inhibitors. A person skilled in the art of treatingGram-negative infections may easily recognize additional, clinicallyrelevant antibacterial agents that may be useful. Preferably saidantibacterial agents are selected from a group of hydrophobic ormoderately hydrophobic antibacterial agents against which the outermembrane of Gram-negative bacteria acts as an effective permeabilitybarrier.

The invention also includes the use of the present compounds orcombinations thereof to sensitize clinically important bacteria listedherein to the host defense mechanism complement (present in the freshhuman and animal serum) by subjecting said bacteria to the action ofsuch compounds during a clinical infection or a suspected infection. Thehost defense can be exerted, e.g., by the combined action of complementand polymorphonuclear leucocytes.

Those skilled in the art of medicine can readily optimize effectivedosages and administration regimens for the compounds according to thepresent invention as well as for the antibiotics in concurrentadministration, taking into account factors well known in the artincluding type of subject being dosed, age, weight, sex and medicalcondition of the subject, the route of administration, the renal andhepatic function of the subject, the desired effect, the particularcompound according to the present invention employed and the toleranceof the subject to it. Dosages of all antimicrobial agents should beadjusted in patients with renal impairment or hepatic insufficiency, dueto the reduced metabolism and/or excretion of the drugs in patients withthese conditions. Doses in children should also be reduced, generallyaccording to body weight.

The total daily dose of a derivative according to the present inventionadministered to a human or an animal can vary, for example, in amountsfrom 0.1 to 100 mg per kg body weight, preferably from 0.25 to 25 mg perkg body weight.

It will also be recognised by one skilled in the art that the optimalcourse of treatment, i.e., the number of doses given per day for adefined number of days, will be determined by the nature and extent ofthe condition being treated, the form, route and site of administration,and the particular patient being treated, and that such optimums can bedetermined by conventional techniques.

There is also provided a method for assaying a compound according to thepresent invention, said compound being a derivative of a naturalpolymyxin or octapeptin, wherein said derivative has a only 2-3 positivecharges, in contrast to the naturally occurring compound from which itis derived, for antibacterial activity against a harmful Gram-negativebacterium and/or for the ability to sensitize it to antibacterial agentsand/or the complement present in the serum, said method comprising thestep of contacting the bacterium with said derivative of a naturalpolymyxin or octapeptin, and identifying derivatives possessingantibacterial activity and/or sensitizing activity towards saidbacterium.

There is also provided a method to screen polymyxin and octapeptinderivatives with reduced binding to renal tissue or its constituents inor from test animals or from human origin measuring their reducedability to competitively block the binding of aminoglycosides to thesame, or block the binding of other substances known to bind to thesame.

In a further aspect there is provided a method for developing novelantibiotics comprising the steps of providing a natural polymyxin oroctapeptin compound, or a derivative thereof, having a total of 4 or 5positive charges, or a total of 6 positive charges, as indeacylpolymyxins, substituting from 1 to 4 residues carrying one or morepositive charges with a residue not having a positive charge, or with acovalent bond, thereby generating a polymyxin derivative having 2 or 3positive charges, assaying said derivative compound for antibacterialactivity against Gram-negative bacteria and/or for the ability tosensitize Gram-negative bacteria to an antibacterial agent, andselecting compounds having antibacterial activity against Gram-negativebacteria, or the ability to sensitize Gram-negative bacteria to anantibacterial agent.

There is also provided in accordance with the present invention asemisynthetic polymyxin derivative obtainable by treating chemically orenzymatically naturally-occurring polymyxins or octapeptins,respectively, or those variants thereof which are manufactured bygenetically modified organisms. Chemical treatments include, but are notlimited to, those with acetanhydride, formic acid, hydrazine, and oxalicacid. Enzymatic treatments include, but are not limited to, with enzymessuch as polymyxin deacylase, ficin, papain, bromelain,subtilopeptidases, subtilisin, colistin hydrolase, and Nagarse.

Preferred compounds according to one embodiment are less cationic thannatural polymyxins or octapeptins, have two (2) or three (3) positivecharges only, and are:

(a) able to inhibit the growth of Escherichia coli, Klebsiellapneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacterfreundii, Pseudomonas aeruginosa or Acinetobacter baumannii and/or tosensitize any of them to antibiotics, and/or

(b) less toxic than clinically used polymyxins, as evidenced in in vivoanimal model, and/or

(c) less nephrotoxic than clinically used polymyxins, as evidenced in ananimal model and/or in an in vitro test that measures affinity of thecompounds to kidney structures, and/or

(d) able to cause less histamine liberation from the tissues thanclinically used polymyxins when administered topically or when inhaledas an aerosol, and/or

(e) pharmacokinetically more favorable, such as having a longer serumhalf life and/or by being less inactivated by polyanionic tissue and pusconstituents than clinically used polymyxins.

Methods for synthesising compounds according to the present inventioninclude but are not limited to the following described below. For aspecific compound to be synthetised, an expert in the art is able tochoose the appropriate method.

1. Semisynthetic derivatives of polymyxins and octapeptins that carry anunchanged heptapeptide part and a modified acyl-aminoacyl side chain canbe made by the procedures described as follows:

Protection of the free amino groups in the starting material (polymyxinor octapeptin, or modifications thereof) by methods known to thoseskilled in the art. The protection can be achieved by the use ofresidues such as t-butoxycarbonyl (tBoc), fluorenylmethoxycarbonyl(Fmoc), benzyloxycarbonyl (CBZ, Z), allyloxycarbonyl (ALOC),3-pyridyl-N-oxide-methoxycarbonyl (as described in patent publication GB1323962), by using Schiff bases such as benzaldehyde by the methoddescribed in Japanese Patent publication 7115630/1971 or the like whichcan be removed by conventional conditions compatible with the nature ofthe product.

In conditions where the poor water solubility occasionally poses aproblem in the sub-sequent steps, the protection can be made by usingnegatively-charged blocking groups such as a sulfonic acid derivative ofFmoc or a carboxylic acid derivative of Fmoc, the method being describedin US patent publication 2006004185. The water solubility can also beenhanced by linking a suitable, removable, negatively charged, veryhydrophilic blocking group to the OH-group of threonine.

Thereafter, the compound is subjected to an enzymatic treatment withenzymes such as polymyxin deacylase, polymyxin hydrolase, papain, ficin,bromelain, subtilopeptidase, Nagarse or other enzymes that remove aterminal part of the side chain or even the entire side chain ofpolymyxin or octapeptin compounds. This treatment can optionally befollowed by the Edman degradation procedure. The resultant compoundlacks the entire side chain and consists of the cyclic heptapeptide partonly, but has a free N-terminal alpha amino group.

Alternatively, polymyxins and octapeptins that have amino groupsprotected by benzyloxycarbonyl can be treated by oxalic acid or formicacid to yield protected deacylderivatives, the method being described byKurihara et al. (1974). The procedure is followed by further enzymetreatment as above and/or by Edman degradation to yield a heptapeptide.

Thereafter, a suitable residue is linked to the free alpha-aminoposition of the heptapeptide ring portion. The residue might contain anacyl or related residue as well as optionally amino acid residues,preferably up to three residues. For instance, one semisyntheticcompound with an acyl group and two amino acid residues can be preparedby adding to the above-described heptapeptide a syntheticN-(acyl)-threonyl-Dthreonyl residue. This can be achieved byconventional general techniques known to those familiar with the art oforganic chemistry, these techniques including the use ofN-hydroxysuccinimide-linked residues as described in US 2006004185. Inthis particular synthesis the procedure may involve the use of2-N-(n-octanoyl)-threonyl-Dthreonyl-N-hydroxysuccinimide.

2. Acylated polymyxin nonapeptides carrying three (3) free amino groups.Polymyxin D possesses only four (4) positive charges. It can be treatedwith papain or ficin by the method described by Chihara et al. (1973)and by Vaara and Vaara (1983a,b) to yield the corresponding nonapeptide.The nonapeptide can then be acylated by acylisotiocyanate (by the methodwell-known to a person skilled in the art and described in US2006004185, by acyl chloride (by the method well-known to a personskilled in the art and described in Chihara et al. 1974), or by usingresidues linked to N-hydroxysuccinimide (by the method well-known to aperson skilled in the art and described in US 2006004185. The acylatedpolymyxin D nonapeptide carries only three (3) free amino groups, all inthe heptapeptide ring portion.

Alternatively, the free amino groups of polymyxin D can be protected bythe means described above. This is followed by an enzymatic treatmentand an optional Edman degradation step, to yield a nonapeptide, whichcan then be acylated by acylisotiocyanate (by the method well-known to aperson skilled in the art and described in US 2006004185, by acylchloride (by the method well-known to a person skilled in the art anddescribed in Chihara et al. 1974), or by using residues linked toN-hydroxysuccinimide (by the method well-known to a person skilled inthe art and described in US 2006004185, Finally, the protective groupsare removed.

In an analogous manner, acylated polymyxin S nonapeptide and acylatedpolymyxin F nonapeptide can be made. Both carry only three (3) freeamino groups.

3. Acylated polymyxin and octapeptin heptapeptides. Heptapeptides can bemade by Nagarse treatment of the natural compounds, as described byKimura et al. 1992. Alternatively, they can be made by treatments withother enzymes such as polymyxin acylase, polymyxin hydrolase, ficin,papain, bromelain, and subtilopeptidase, followed by optional Edmandegradation steps. They can also be made by deacylating the naturalcompounds by hydrazine or by acids such as formic acid and oxalic acid,followed by Edman degradation steps. The heptapeptide can then beacylated for instance by using the acyl chloride technique well-known toa person skilled in the art and described in Chihara et al. (1974). Theacylated polymyxin heptapeptide carries only three (3) free aminogroups.

4. Totally synthetic polymyxin and octapeptin derivatives can be made bythe very conventional methods known for those skilled in the art. Suchmethods include the liquid-phase synthesis procedures as well as thesolid-phase synthesis procedures described for instance by Sakura et al.(2004), Tsubery et al. (2000a, 2000b, 2002, 2005), and Ofek et al.(2004). The methods include e.g. the use of protecting agents such asFmoc, tBoc, and CBZ at strategic positions, as well as the cyclisationstep where DPPA (diphenyl phosphorazidate) or a mixture ofbenzotrizole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBop), N-hydroxybenzotriazole (HoBt), and N-methylmorpholine (NMM) isused. Fmoc derivatives of many non-trivial as well as D-amino acids arecommercially available.

5. Exemplary reactions related to conversion of the free amino groups togenerate compounds according to the present invention having 2 or 3positive charges may include (but are not limited to) the followingreactions:

A) reaction of free amine group of the compound with a conjugationmoiety comprising a reactive epoxide group, thereby generating aβ-hydroxyamine linkage;

B) reaction of free amine group of the compound with a conjugationmoiety comprising a reactive sulphonyl halide, thereby generating asulfonamide linkage;

C) reaction of free amine group of the compound with a conjugationmoiety comprising a reactive carboxyl acid, thereby generating an aminelinkage;

D) reaction of free amine group of the compound with a conjugationmoiety comprising a reactive aldehyde group (under reducing conditions),thereby generating an amine linkage;

E) reaction of free amine group of the compound with a conjugationmoiety comprising a reactive ketone group (under reducing conditions),thereby generating an amine linkage;

F) reaction of free amine group of the compound with a conjugationmoiety comprising a reactive isocyanate group, thereby generating a urealinkage.

LIST OF REFERENCES

-   All references cited in the present application are hereby    incorporated by reference in their entirety.-   Chihara S, Tobita T, Yahata M, Ito A, Koyama Y. 1973. Enzymatic    degradation of colistin. Isolation and identification of α-N-Acyl    α,γ-diaminobutyric acid and colistin nonapeptide. Agr Biol Chem    37:2455-2463.-   Chihara S, Ito A, Yahata M, Tobita T, Koyama Y. 1974. Chemical    synthesis, isolation and characterization of α-N-fattyacyl colistin    nonapeptide with special reference to the correlation between    antimicrobial activity and carbon number of fattyacyl moiety. Agric    Biol Chem 38:521-529.-   Kimura Y, Matsunaga H, Vaara M. 1992. Polymyxin B octapeptide and    polymyxin B heptapeptide are potent outer membrane    permeability-increasing agents. J Antibiot 45:742-749.-   Kurihara T, Takeda H, Ito H, Sato H, Shimizu M, Kurosawa A. 1974.    Studies on the compounds related to colistin. IX. On the chemical    deacylation of colistin and colistin derivatives. Yakugaku Zasshi    94:1491-1494.-   Nagai J, Saito M, Adachi Y, Yumoto R, Takano M. 2006. Inhibition of    gentamicin binding to rat renal brush-border membrane by megalin    ligands and basic peptides. J Control Release 112:43-50.-   Nikaido H. 2003. Molecular basis of bacterial outer membrane    permeability revisited. Microbiol. Molec Biol Rev 67:593-656.-   Nikaido H, Vaara M. 1985. Molecular basis of bacterial outer    membrane permeability. Microbiol. Rev 49:1-32.-   Rose F, Heuer K U, Sibelius U, Hombach-Klonisch S, Ladislau K,    Seeger W, Grimminger F. 1999. Targeting lipopolysaccharides by the    non-toxic polymyxin B nonapeptide sensitizes resistant E. coli to    the bactericidal effect of human neutrophils. J Infect Dis    182:191-199.-   Sakura N, Itoh T, Uchida Y, Ohki K, Okimura K, Chiba K, Sato Y,    Sawanishi H. 2004. The contribution of the N-terminal structure of    polymyxin B peptides to antimicrobial and lipopolysaccharide binding    activity. Bull Chem Soc Jpn 77:1915-1924.-   Srinivasa B D, Ramachandran L K. 1978. Chemical modification of    peptide antibiotics: Part VI-Biological activity of derivatives of    polymyxin B. Ind J Biochem Biophys 14:54-58.-   Srinivasa B D, Ramachandran L K. 1979. The polymyxins. J Scient    Industr Res 38:695-709.-   Srinivasa B D, Ramachandran L K. 1980. Essential amino groups of    polymyxin B. Ind J Biochem Biophys 17:112-118.-   Storm D R, Rosenthal K S, Swanson P E. 1977. Polymyxin and related    peptide antibiotics. Annu Rev Biochem 46:723-63.-   Teuber M. 1970. Preparation of biologically active    mono-N-acetyl(14C)-derivatives of the membrane-specific polypeptide    antibiotic polymyxin B. Z Naturforsch 25b:117.-   Tsubery H, Ofek I, Cohen S, Fridkin M. 2000a. Structure-function    studies of polymyxin B nonapeptide: Implications to sensitization of    Gram-negative bacteria. J. Med Chem 43:3085-3092.-   Tsubery H, Ofek I, Cohen S, Fridkin M. 2000b. The functional    association of polymyxin B with bacterial lipopolysaccharide is    stereospecific: Studies on polymyxin B nonapeptide. Biochemistry    39:11837-11844.-   Tsubery H, Ofek I, Cohen S, Fridkin M. 2001. N-terminal    modifications of polymyxin B nonapeptide and their effect on    antibacterial activity. Peptides 22:1675-1681.-   Tsubery H, Ofek I, Cohen S, Eisenstein M, Fridkin M. 2002.    Modulation of the hydro-phobic domain of polymyxin B nonapeptide:    effect on outer-membrane permeabilization and lipopolysaccharide    neutralization. Molecular Pharmacology 62:1036-42.-   Tsubery H, Yaakov H, Cohen S, Giterman T, Matityahou A, Fridkin M,    Ofek I. 2005. Neopeptide antibiotics that function as opsonins and    membrane-permeabilizing agents for gram-negative bacteria.    Antimicrob Agents Chemother 49:3122-3128.-   Vaara M. 1992. Agents that increase the permeability of the outer    membrane. Microbiol. Rev 56:395-411.-   Vaara M. 1993. Antibiotic-supersusceptible mutants of Escherichia    coli and Salmonella typhimurium. Antimicrob Agents Chemother    37:2255-2260.-   Vaara M, Vaara T. 1983a. Sensitization of Gram-negative bacteria to    antibiotics and complement by a nontoxic oligopeptide. Nature    (London) 303:526-528.-   Vaara M, Vaara T. 1983b. Polycations sensitize enteric bacteria to    antibiotics. Antimicrob Agents Chemother 24:107-113.-   Vaara M, Vaara T. 1983c. Polycations as outer membrane-disorganizing    agents. Antimicrob Agents Chemother 24:114-122.-   Vaara M, Viljanen P, Vaara T, Makela P. 1984. An outer membrane    disorganizing peptide PMBN sensitizes E. coli strains to serum    bactericidal action. J Immunol 132:2582-2589.-   Viljanen P, Matsunaga H, Kimura Y, Vaara M. 1991. The outer membrane    permeability-increasing action of deacylpolymyxins. J Antibiotics    44:517-523.

EXAMPLES

The following examples illustrate certain embodiments of the presentinvention and should not be construed as limiting the scope of theinvention.

Example 1 Peptide Synthesis

Polymyxin derivatives (“NAB peptides” or “NAB compounds”) weresynthesized by conventional solid phase chemistry, using the standardFmoc protection strategy. The amino acid at the C-terminus iscommercially available as pre-attached to the solid phase and whencleaved off the resin with acid, yields a C-terminal carboxylic acid.

The strategy in the protection was to use three levels of orthogonalprotection, temporary Fmoc protection for the alpha amino functions,groups which are removed during the acid cleavage stage, andsemi-permanent protection to cover reactive side chain functions whilethe cyclisation reaction takes place. After cleavage of the peptide fromthe resin, the C-terminal carboxylic acid is reacted with an aminofunction on the side chain of one of the amino acids to form a cyclicpeptide. After the cyclisation step, the semi-permanent protectiongroups are removed to yield NAB peptide.

Accordingly, the alpha amino function of the amino acid was protected byfluorenyl-methoxycarbonyl (Fmoc) and Fmoc was removed by 20% piperidinein DMF at every cycle. The amino acid that is involved with cyclisation,e.g. diaminobutyric acid, was protected by t-butoxycarbonyl (tBoc), anacid labile group which was removed at the cleavage step. All the otheramino acids which have functional side chain groups were protected by agroup that is stable to the acid cleavage stage, i.e. benzyloxycarbonyl(Z). Amino acids phenylalanine and leucine naturally needed no sidechain protection. The amino terminus was not protected; this enableddirect reaction in the acylation procedure.

The synthesis steps were performed in a commercial automatizedsyntesizer that employed0-(6-Chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HCTU) as activator.

6-methylheptanoic acid (6-MHA) was from Ultra Scientific Inc, NorthKingstown, R1, USA (product number, FLBA 002). Other fatty acids werefrom a standard supplier.

The acylation was performed by using a four-fold molar excess of eachamino acid or the fatty acid, four-fold molar excess of the activatorHCTU (see above), and an eight-fold molar excess of N-methyl morpholine.The reaction time was 30 min.

The amino acids were purchased already protected from a standardsupplier. The peptide was removed from the resin by reaction with asolution of 95% trifluoroacetic acid and 5% water for 2 hours at roomtemperature, to yield the partially protected product. The resultingpeptide was precipitated with diethyl ether.

The cyclisation mixture used wasbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBop), N-hydroxybenzotri-azole (HoBt), and N-methyl morpholine (NMM)at the molar excess of 2, 2, and 4, respectively. The peptide wasdissolved in dimethylformamide, the cyclisation mix was added andallowed to react for 2 hours. The cyclised, protected peptide wasprecipitated by the addition of cold diethyl ether. Any residual PyBopwas removed by washing the peptide with water.

The remaining side chain protection groups (Z) were removed by catalyticdehydrogenation. The peptide was dissolved in acetic acid-methanol-water(5:4:1), under an atmosphere of hydrogen and in the presence of apalladium charcoal catalyst.

The peptide was purified by reverse phase chromatography usingconventional gradients of acetonitrile:water:trifluoroacetic acid. Theproduct was dried by lyophilisation.

The yield was 20-40 mg representing approx. 20%-40% of the theoretical,calculated from the molar amount (approx. 100 micromoles) of the firstamino acyl residue bound to the resin.

The purity, as estimated by reversed phase HPLC was more than 95%. Foracylated peptides, the Edman degradation product did not reveal anyamino acid residue, indicating that the α-amino group of the N-terminalamino acid residue was blocked, as expected, due to the successfulN-acylation. Within experimental error, the masses obtained were thoseexpected from the theoretical values.

Example 2 Direct Antibacterial Activity of the Compounds AgainstEscherichia coli

Peptides synthesized in Example 1, all carrying at least two (2) but notmore than three (3) positive charges, were studied for their ability toinhibit the growth of E. coli. This was tested employing LB agar (LBAgar Lennox, Difco, BD, Sparks, Md., U.S.A) plates. The indicatororganism E. coli IH3080 (K1:O18) was an encapsulated strain originallyisolated from a neonate suffering from meningitis (Vaara et al. 1984)and obtained from National Public Health Institute, Helsinki, Finland.

From an overnight-grown culture of IH3080 on LB agar, a suspension ofapprox. 10⁸ cells/ml was prepared in 0.9% NaCl. Aliquots of thissuspension were then pipetted on the agar plates and the plates weregently shaken to spread the suspension evenly on the entire surface ofthe plate. Thereafter, the unabsorbed part of the suspension was removedby using a Pasteur pipette. After the surface had dried, small wells(diameter, 2 mm) were drilled on the plates (five wells per plate) byusing a sterile sharp-edged narrow metal tube, single-use pipette tip,and vacuum suction. Samples (4 μl and 10 μA of the peptide solution in0.9% NaCl (at concentrations of 1 μg/ml and 0.1 μg/ml) were thenpipetted to the wells and the sample fluids were allowed to absorb.Controls included 0.9% NaCl solution without the compound to be tested.The plates were then incubated for 18 h at 37° C. whereafter thediameters of growth inhibition zones around each well were measured; thediameter of the well itself was not reduced. Finally, the diameters wereconverted to surface areas of growth inhibition (in square mm).

Table 2 shows the antibacterial activity of the derivatives against E.coli IH3080 as compared with that of an equal amount of polymyxin B aswell as of some polymyxin derivatives not related to the presentinvention. NAB734, NAB737, NAB739 and NAB740 were the most antibacterialcompounds and were even more antibacterial than polymyxin B against E.coli IH3080. A well containing 4 μg of NAB739 produced a growthinhibition area as wide as 133 square mm. In all these four NABcompounds, the side chain consists of two amino acyl residues that carryhydroxyl groups.

Unlike NAB739, NAB7061 was not antibacterial at 4 μg. However, itdisplayed notable antibacterial activity at 10 μg. NAB7061 differs fromNAB739 only by carrying Abu (instead of DSer) in R3. Prolonging thelength of the fatty acyl part from C8 in NAB7061 to C10 in NAB7062resulted in notably enhanced antibacterial activity manifesting at 4rig. In addition, three other peptides (NAB738, NAB716 and NAB719)displayed notable antibacterial activity, albeit clearly weaker thanNAB739 and the other most antibacterial compounds.

A common property of the compounds directly antibacterial to E. coli wasthe presence of three positive charges out of which either all the threeor at least two were located in suitable positions in the cyclic part.In the latter case the relative positions of said charges significantlyaffected the potency of the antibacterial activity against E. coli.

Furthermore, as shown in the Table 2, also the structure and length ofthe side chain has significant influence on the potency of theantibacterial activity. The presence of a side chain consisting of atleast two amino acyl residues appears to be important for the compoundsantibacterial against E. coli, since the compounds lacking either R2(NAB713) or both R2 and R3 (octanoyl PBHP) also lacked the directantibacterial activity in the conditions used in the assay. However, itcan be anticipated, that the lack of those residues can be compensatedby using instead of octanoyl residue, a more extended residue as theR(FA).

TABLE 2 Structure of the compounds and their antibacterial activity* against Escherichia coli IH3080 Structure** Positive AntibacterialPeptide sequence*** changes in activity at side cyclic SEQ ID cyclicFA-part chain part No. part total 4 μg 10 μg Reference compoundsPolymyxin B MO(H)A XTX cy[XX FLXXT] 1 3 5 79 133 Colistin (polymyxin E)MO(H)A XTX cy[XX LLXXT] 2 3 5 ND ND Deacylpolymyxin B — +XTX cy[XXFLXXT] 3 3 6 57 113 Deacylcolistin — +XTX cy[XX LLXXT] 4 3 6 79 95Polymyxin B nonapeptide — +TX cy[XX FLXXT] 5 3 5 0 0Polymyxin B heptapeptide — + cy[XX FLXXT] 6 4 4 0 0 NAB704 — +TZ cy[XXFLXXT] 7 3 4 0 0 NAB705 — +ZTZ cy[XX FLXXT] 8 3 4 0 0 Octanoyl PMBH OA —cy[XX FLXXT] 9 3 3 0 0 Cpds of the present invention NAB 739 OA TS cy[XXFLXXT] 10 3 3 133 177 NAB 740 DA TS cy[XX FLXXT] 10 3 3 95 133 NAB 737OA TT cy[XX FTXXT] 11 3 3 133 201 NAB 734 OA TS cy[XX FTXXT] 12 3 3 113177 NAB 7062 DA TZ cy[XX FLXXT] 13 3 3 50 99 NAB 7061 OA TZ cy[XX FLXXT]13 3 3 0 50 NAB 706 MHA TZ cy[XX FLXXT] 13 3 3 5 7 NAB 707 MHA ZTZ cy[XXFLXXT] 14 3 3 5 7 NAB 716 OA TX cy[XX FLZXT] 15 2 3 13 64 NAB 719 OA TZcy[XX FLXXL] 16 3 3 7 50 NAB 738 OA TA cy[XX FTXXT] 17 3 3 0 64 NAB 717OA TX cy[XX FLXZT] 18 2 3 0 0 NAB 718 OA TZ cy[XX LLXXT] 19 3 3 0 0NAB 733 OA AA cy[XX FLXXT] 20 3 3 0 0 NAB 736 DA — cy[XX FLXXT] 9 3 3 00 NAB 713 OA Z cy[XX FLXXT] 21 3 3 0 0 NAB 715 OA TX cy[XZFLXXT] 22 2 30 0 NAB 721 MHA XTX cy[XX FLZZT] 23 1 3 0 0 NAB 731 OA TZ cy[XK FLXXT]24 3 3 0 0 NAB 710 OA TZ cy[XZFLXXT] 25 2 2 0 7 NAB 709 OA TZ cy[XXFLXZT] 26 2 2 0 0 NAB 708 OA TZ cy[XX FLZXT] 27 2 2 0 0 NAB 725 OA XTXcy[XZFLXZT] 28 1 3 0 0 NAB 726 OA XTX cy[XZFLZXT] 29 1 3 0 0 NAB 722 MHAXTX cy[XZFLZZT] 30 0 2 0 0 NAB 735 OA XXX cy[XZFLZZT] 31 0 3 0 0 NAB 701— +TX cy[XX FLZZT] 32 1 3 0 0 NAB 702 — +TX cy[XX FLBBT] 33 1 3 0 0NAB 703 — +TX cy[XX FLJJT] 34 1 3 0 0 * Antibacterial activity measuredas the growth inhibition (in square millimeters) around a wellcontaining 4 or 10 microgram of a compound on LB plates ** One-lettercodes for amino acyl residues: A, Ala; F, Phe; K, Lys; L,Leu; S, Ser; T,Thr; X Dab; Z, Abu; B, N-g-formyl-Dab; J, N-g-acetyl-Dab. Underlinedletters indicate residues that are in D-configuration. Bold lettersindicate residues that carry a positive charge. Bold + indicates thepositive charge of the +60- amino group in the free N-terminus of thepeptide. Abbreviation: cy, cyclo. *** In the sequence listing X, Z, Band J are denoted Xaa, and defined as modified residues (MOD_RES).

Example 3 Direct Antibacterial Activity of Selected Nab CompoundsAgainst Acinetobacter baumannii and Pseudomonas aeruginosa

Direct antibacterial activity of twelve NAB compounds againstAcinetobacter baumannii ATCC 19606 and Pseudomonas aeruginosa ATCC 27853was tested by using the susceptibility determination method described inExample 2. The results are shown in Table 3. Five compounds (NAB7062,NAB734, NAB737, NAB739, and NAB740) had notable activity against A.baumannii. In Example 2, the same compounds were shown to be very potentagainst E. coli. The antibacterial activity of NAB739 and NAB740 were asstrong as or even stronger than that of polymyxin B.

Against P. aeruginosa, the most active NAB compounds were NAB739, NAB740as well as NAB736, which is quite inactive against E. coli andAcinetobacter baumannii. NAB740 was the most active compound and itsactivity was as strong as that of polymyxin B. All the three NABcompounds lack positive charges in the side chain and were still activeagainst P. aeruginosa. This finding is against the conclusion ofSrinivasa and Ramachandran (1980a) that the free amino groups in R1 andR3 are essential for the growth inhibition of P. aeruginosa.

Surprisingly, NAB736 is rather effective against P. aeruginosa whereasoctanoyl PMBH is much less effective. Accordingly, lengthening the R(FA)part from C8 to C10 has a marked effect on the activity.

TABLE 3 Antibacterial activity* of twelve (12) novel compounds againstAcinetobacter baumannii and Pseudomonas aeruginosa A. baumannii Ps.aeruginosa ATCC 19606 ATCC 27853 4 μg 10 μg 4 μg 10 μg NAB 7061 0 0 0 0NAB 7062 13 38 0 0 NAB 716 0 0 0 0 NAB 717 0 0 0 0 NAB 718 0 0 0 0 NAB719 0 0 0 0 NAB 736 0 0 38 79 NAB 734 38 95 13 50 NAB 737 38 79 0 28 NAB738 0 0 0 0 NAB 739 113 177 38 79 NAB 740 133 201 64 133 Polymyxin B 113154 95 133 PMBN 0 0 113 177 *Antibacterial activity measured as thegrowth inhibition (in square millimeters) around a well containing 4 or10 μg of the compound on LB plates

Example 4 Direct Antibacterial Activity of NAB734 Against SelectedGram-Negative Bacteria

The susceptibility of eleven Gram-negative bacterial strains (ninedifferent species) to NAB734 and polymyxin B was compared by using thesusceptibility determination method described in Example 2. The strainsincluded those belonging to the species of Serratia marcescens andProteus mirabilis, both species generally known to be resistant topolymyxin. Furthermore, the susceptibility determination was alsoperformed by using the Gram-positive bacterium, Staphylococcus aureus,also generally known to be polymyxin-resistant. Ten of the strainsoriginated from ATCC (American Type Culture Collection, Manassas, Va.,U.S.A.), and one from CCUG (Culture Collection of University ofGothenburg, Sweden). The source of E. coli IH3080 has been described inExample 2. Polymyxin B sulfate was from Sigma-Aldrich (St. Louis, Mo.,USA).

The results in Table 4 show that NAB734 can be generally regarded to beapproximately as potent as polymyxin B against E. coli, Klebsiellapneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Citrobacterfreundii. It appears to be somewhat less potent than polymyxin B againstAcinetobacter baumannii and clearly less potent than polymyxin B againstPseudomonas aeruginosa. Representatives of known polymyxin-resistantbacterial species were resistant to NAB734 also. This suggests thatNAB734 has a very specific antibacterial action and that its mode ofaction is quite similar to that of polymyxin B.

TABLE 4 Antibacterial activity* of NAB 734 against selectedGram-negative bacteria and Staphylococcus aureus Polymyxin NAB 734 Bsulfate Strain 4 μg 10 μg 4 μg 10 μg E. coli ATCC25922 133 177 95 133 E.coli IH3080 113 154 95 133 K. pneumoniae ATCC13883 64 113 79 113 K.pneumoniae CCUG45421 64 104 95 133 K. oxytoca ATCC13182 95 143 79 104 E.cloacae ATCC23355 133 177 95 143 C. freundii ATCC8090 133 177 95 154 A.baumannii ATCC19606 57 79 113 154 P. aeruginosa ATCC27853 13 50 95 133S. marcescens ATCC8100 0 0 0 0 P. mirabilis ATCC29906 0 0 0 0 S. aureusATCC 25923 0 0 0 0 *Antibacterial activity measured as the growthinhibition (in square millimeters) around wells containing 4 or 10 μg ofthe compound on LB plates

Example 5 The Ability of the Nab Compounds to Sensitize E. Coli IH3080to a Model Antibiotic Rifampin

Novel NAB peptides according to the present invention and all carryingat least two (2) but not more than three (3) positive charges, were alsostudied for their ability to sensitize E. coli IH3080 to rifampin. Thiswas tested in parallel with the susceptibility determinations describedin Example 2 and by employing LB plates that contain increasingconcentrations (0.1 μg/ml, 0.3 μg/ml, 1 μg/ml) of rifampin(Sigma-Aldrich, St. Louis, Mo., U.S.A).

Table 5 shows the activity of the NAB compounds (4 μg) against E. coliIH3080 in the presence of rifampin (0.1 and 1 μg/ml) as compared withthe activity of an equal amount of previously described substances knownto sensitize Gram-negative bacteria to antibacterial agents, i.e.polymyxin B heptapeptide, deacylpolymyxin B, deacylcolistin, polymyxin Bnonapeptide, as well as with polymyxin B. The compounds also includedoctanoyl PMBH, an agent that has not previously been reported to be ableto sensitize bacteria to antibiotics.

Several NAB compounds sensitized E. coli IH3080 to the antibacterialeffect of as low a concentration of rifampin as 0.1 μg/ml. In theabsence of the compounds tested, a hundred-fold concentration (10 μg/ml)of rifampin was needed for the inhibitory effect. Several compounds thatlacked a notable direct antibacterial activity at 4 μg were able tosensitize the target bacterium to rifampin. Such compounds includedNAB7061, NAB717, NAB718, and NAB733.

Furthermore, most of the NAB compounds that had direct antibacterialactivity, i.e. antibacterial activity in the absence of rifampin (seeExample 2), inhibited the target bacterium even more effectively in thepresence of rifampin. The ability of the most active compounds NAB734,NAB737, NAB738, and NAB739 was clearly even better than that of PMBN,the well-known effective permeabilizer of the OM.

The most active NAB compounds carry three (3) positive charges and haveat least two (2) positive charges suitably positioned in the cyclicpart, the relative positioning of which affecting the potency of thesensitizing activity. It should indeed be noted, that NAB716, thatcarries only two positive charges in the cyclic part and has the thirdpositive charge in the form of a Dab residue in R3, is notably able tosensitize E. coli to rifampin.

Amongst the series of compounds all having octanoyl residue as R(FA),octanoyl PMHP, that lacks both R2 and R3, possessed the weakestsensitizing activity, NAB713, that lacks R2, possessed a somewhat betteractivity, and NAB7061, that possesses both R2 and R3 had a remarkableactivity. This indicates that the presence of R2 and R3 is advantageous.However, their absence can be at least partially compensated bylengthening the R(FA) part as in NAB736. It carries decanoyl residue asthe R(FA) part, lacks both R2 and R3, and is quite active as asensitizer.

NAB compounds that carry both of their two (2) positive charges in thecyclic part were less active than the structurally otherwise analogousNAB compounds that carry all of their three (3) positive charges in thecyclic part, or, for one compound (NAB708), inactive in the studyconditions employed.

NAB compounds that carry two (2) positive charges in the side chain andone (1) positive charge in the cyclic part have very modest, if anyactivity, in the study conditions employed. NAB735, that carries all ofits three (3) positive charges in the side chain is inactive in thestudy conditions employed. Again the relative positions of the saidcharges in the cyclic part affected the potency of the sensitizingactivity.

TABLE 5 Antibacterial activity of the compounds (4 μg) against E. coliIH3080 in the presence of rifampin* Antibacterial activity in thepresence of rifampin concn (μg/ml) of 0 0.1 1.0 Reference and othercompounds Polymyxin B 79 95 104 Colistin (polymyxin E) ND ND NDDeacylpolymyxin B 57 79 127 Deacylcolistin 79 87 127 Polymyxin Bnonapeptid

0 20 113 Polymyxin B heptapepti

0 0 38 NAB 704 0 0 24 NAB 705 0 0 5 Octanoyl PMBH 0 0 38 Novel compoundsof the present invention NAB 739 133 177 201 NAB 740 95 95 95 NAB 737133 177 201 NAB 734 113 154 201 NAB 7062 50 104 104 NAB 7061 0 113 155NAB 706 5 79 133 NAB 707 5 87 113 NAB 716 13 133 165 NAB 719 7 79 95 NAB738 0 133 177 NAB 717 0 71 95 NAB 718 0 13 133 NAB 733 0 95 113 NAB 7360 113 133 NAB 713 0 20 38 NAB 715 0 0 33 NAB 721 0 13 28 NAB 731 0 0 5(22*) NAB 710 0 7 13 NAB 709 0 0 13 NAB 708 0 0 0 NAB 725 0 0 0 NAB 7260 0 0 NAB 722 0 0 0 NAB 735 0 0 0 NAB 701 0 0 0 NAB 702 0 0 0 NAB 703 00 0 *Antibacterial activity measured as the growth inhibition (in squaremillimeters) around a well containing 4 μg of a compound on plates withno rifampin or rifampin (0.1 or 1.0 μg/ml) **The value in parentheseswas obtained using a well containing 10 μg of the compound

indicates data missing or illegible when filed

Example 6 The Ability of the Nab Compounds to Sensitize Acinetobacterbaumannii And Pseudomonas aeruginosa to a Model Antibiotic Rifampin

NAB peptides related to present invention were also studied for theirability to sensitize A. baumannii and P. aeruginosa to rifampin (Table6). This was tested in parallel with the susceptibility determinationsdescribed in Example 3 and by employing LB plates that containincreasing concentrations (0.1 μg/ml, 0.3 μg/ml, 1 μg/ml) of rifampin.

Several NAB compounds possessed a very marked ability to sensitize A.baumannii to rifampin. The ability of the most active compounds NAB734,NAB737, and NAB739 was clearly even better than that of PMBN, thewell-known effective permeabilizer of the OM. NAB739 inhibited thegrowth of P. aeruginosa somewhat better in the presence of rifampin thanin its absence.

TABLE 6 Antibacterial activity* of twelve (12) novel compounds (4 μg)against Acinetobacter baumannii and Pseudomonas aeruginosa in thepresence of rifampin (0.1 or 0.3 μg/ml) A. baumannii Ps. aeruginosa ATCC19606 ATCC 27853 0 0.1 0.3 0 0.1 0.3 NAB 7061 0 28 50 0 0 0 NAB 7062 1350 95 0 0 7 NAB 716 0 0 64 0 0 0 NAB 717 0 0 50 0 0 0 NAB 718 0 0 38 0 00 NAB 719 0 154 133 0 0 7 NAB 736 0 95 154 38 38 38 NAB 734 38 201 28313 20 20 NAB 737 38 201 283 0 20 20 NAB 738 0 95 154 0 13 13 NAB 739 113177 314 38 64 64 NAB 740 133 154 154 64 64 64 PMBN 0 133 154 113 113 154*Antibacterial activity measured as the growth inhibition (in squaremillimeters) around a well containing 4 μg of a compound on plates withno rifampin (control) or with rifampin (0.1 or 0.3 μg/ml)

Example 7 NAB7061 Sensitizes E. coli, Klebsiella pneumoniae, andEnterobacter cloacae to a Broad Range of Antibacterial Agents

The minimum inhibitory concentrations (MIC) of a representative set ofclinically used antimicrobial agents were determined for two strains ofE. coli (ATCC25922 and IH3080), K. pneumoniae ATCC13883, and E. cloacaeATCC23355 by using Mueller-Hinton agar medium (product no Lab039; LabMLtd., Bury, Lancs, U.K.) in the presence of NAB7061 (4 μg/ml) as well asin its absence. MICS were determined by using E-strips (Biodisk Ltd.,Solna, Sweden) according to the manufacturer's instructions. The NAB7061concentration used did not itself inhibit the growth of the targetbacteria. The MIC of NAB7061 for E. coli IH3080 and K. pneumoniaeATCC13883 was >16 μg/ml, for E. coli ATCC25922 16 μg/ml, and for E.cloacae ATCC23355 8 μg/ml.

The results are shown in Table 7. NAB7061 at a concentration of 4 μg/mlwas able to sensitize the tested strains to rifampin by a factor rangingfrom 170 to 1500. Sensitization factor is defined as the ratio of theMIC of an antibiotic in the absence of NAB7061 to that in the presenceof 4 μg/ml of NAB7061. Extremely high sensitization factors wereobserved also to clarithromycin (63-380), mupirocin (24-512),azithromycin (31-94), erythromyxin (21-48), and for some of the strains,to fusidic acid, quinupristin-dalfopristin, clindamycin, linezolid, andvancomycin. All these antibacterial agents are notably hydrophobic orlarge (vancomycin) and are known to be excluded by the intact OM ofGram-negative bacteria but penetrate the damaged OM. No significantsensitization (sensitization factor <2, tested by using E. coliATCC25922) was found to piperacillin, ceftazidime, cefotaxime,levofloxacin, ciprofloxacin, meropenem, and tobramycin, all agents thatare hydrophilic or relatively hydrophilic and against which the intactOM is not an effective permeability barrier.

TABLE 7 Sensitization factors* to selected antibacterial agents at NAB7061 concentration of 4 μg/ml E. coli K. pneum. E. cloacae ATCC E. coliATCC ATCC 25922 IH 3080 13883 23355 Rifampin** 250-750 170-350 250-500 750-1500 Clarithromycin*** 170-380 190-260 63  85-380 Mupirocin*** 64-170 64-85 24-32 250-512 Azithromycin*** 24-64 31-94 31-43 32Erythromycin*** 32-48 32-42 24-48 21-43 Fusidic acid >43 >43 >4 >8Quinupristin-dalfopr. >21 >21 1 >5 Clindamycin 6 12 43 32Linezolid >11 >8 >4 >8 Vancomycin 5 2.5 1 32 Polymyxin B 5 2 1 4Trimetoprim 4 3 2 3 Moxifloxacin 2.5 4 1.4 4 *Sensitization factor isthe ratio of the MIC of the antibiotic in the absence of NAB 7061 tothat in the presence of 4 μg/ml of NAB 7061 ** Results from fiveindependent determinations *** Results from two independentdeterminations

Example 8 Susceptibility of 33 Different Strains of Gram-NegativeBacteria to Rifampin and Clarithromycin in the Presence of NAB7061 (4μg/ml)

The minimum inhibitory concentrations (MIC) of rifampin andclarithromycin for a representative set of different strains ofclinically relevant Gram-negative bacteria were determined by the E-testmethod as in Example 7 and by using Mueller-Hinton agar with or withoutNAB7061 (4 μg/ml). This concentration of NAB7061 did not itself inhibitthe growth of the target bacteria. The strains originated from ATCC (11strains), CCUG (11 strains), and NCTC (The National Collection of TypeCultures, Colindale, U.K.; 2 strains). Eight strains (the F-strains)were purchased from Mobidiag Ltd., Helsinki, Finland. The source of E.coli IH3080 has been given in Example 2. Sensitization factor wasdefined as in Example 7.

The results are shown in Table 8. For all strains (17) belonging to thegroup consisting of E. coli, K. oxytoca, E. cloacae, and C. freundii,the MIC of rifampin was as low ≦0.125 μg/ml in the presence of NAB7061(4 μg/ml) and the sensitization factor varied from 85 to 2000. Verysimilar results were obtained with clarithromycin. For fifteen out ofseventeen strains belonging to the group consisting of E. coli, K.oxytoca, E. cloacae, and C. freundii, the MIC of clarithromycin was aslow as ≦0.25 μg/ml in the presence of NAB7061 (4 μg/ml) and for all the17 strains the sensitization factor varied from 90 to 1000. Strains ofK. pneumoniae remained somewhat more resistant to both antibiotics, andthe sensitization factors varied between 10 and 500. For the threestrains of A. baumannii, the sensitization factors varied between 24 and125, and the resulting MIC values were quite low (to rifampin ≦0.125μg/ml and to clarithromycin ≦0.5 μg/ml).

TABLE 8 The ability of NAB 7061 to sensitize Gram-negative bacteria tomodel antibiotics (rifampin and clarithromycin) MIC (μg/ml) of MIC(μg/ml) of rifampin in the Sensitization clarithromycin in theSensitization presence of 4 μg/ml factor** presence of 4 μg/ml factor***Bacterial strain of NAB 7061* to rifampin of NAB 7061 to clarithromycinE. coli ATCC25922**** 0.016-0.047 250-750 0.094-0.125 170-400 E. coliIH3080**** 0.023-0.047 170-350 0.047-0.064 190-260 E. coli CCUG414210.032-0.064 125 0.125 400 E. coli CCUG41422 0.094-0.125 170-256 0.25 100E. coli CCUG41424 0.064-0.094 85-94 0.125 130 E. coli CCUG414250.016-0.023 200-260 0.047 340 E. coli CCUG41427 0.032-0.064 100-2500.064 250 E. coli CCUG41429 0.032 125-250 0.064 250 E. coli CCUG414320.032 180-250 0.064 90 E. coli NCTC13351 0.032-0.047 340-500 0.032 750E. coli NCTC13353 0.064 125-250 1 260 K. pneumoniae ATCC13883****0.064-0.125 250-500 0.19 60 K. pneumoniae CCUG45421 2-3 10-20 24 10 K.pneumoniae F145 0.19-0.25 170 0.25 170 K. pneumoniae F144 0.19 >170 0.7564 K. pneumoniae F136 0.75 >43 2 24 K. oxytoca ATCC13182 0.032-0.047680-750 0.25 260 K. oxytoca CCUG51683 0.012-0.023  700-2000 0.19 250 E.cloacae ATCC23355**** 0.008-0.016  750-1500 0.25-0.75  90-400 E. cloacaeCCUG52947 0.032-0.047  500-1000 0.38 350 E. cloacae F230 0.016-0.0321500 0.047 1000 E. cloacae F232 0.023 1400 0.094 500 C. freundiiATCC8090 0.023-0.032  500-1000 0.125 250 Ac. baumannii ATCC196060.094-0.125 24-32 0.5 50 Ac. baumannii F263***** 0.032-0.19  21-125 0.3840 Ac. baumannii F264 0.125 32 0.25 100 S. maltophilia ATCC17444 2 4-864 4 S. marcescens ATCC8100 16 <2 96 <2 P. mirabilis ATCC29906 1-6 <2 24<2 P. vulgaris ATCC13315   1-1.5 <2 24 <2 P. aeruginosa ATCC27853 12-162 32 <2 P. aeruginosa CCUG51971 >32 <2 64 <2 P. aeruginosa F58 >32 <2256 <2 *Results from two independent determinations **Sensitizationfactor is the ratio of rifampin MIC in the absence of NAB 7061 to thatin the presence of 4 μg/ml of NAB 7061 ***Sensitization factor is theratio of clarithromycin MIC in the absence of NAB 7061 to that in thepresence of 4 μg/ml of NAB 7061 ****Results from five (rifampin) and two(clarithromycin) independent determinations *****Results from threeindependent determinations (rifampin)

Example 9 NAB7061 Sensitizes Carbapenem-Resistant Strains ofAcinetobacter to Carbapenems

The minimum inhibitory concentrations (MIC) of two carpapenems, imipenemand meropenem, for three strains of A. baumannii were determined by theE-test method as in Example 7 and by using Mueller-Hinton agar with orwithout NAB7061 (4 μg/ml). This concentration of NAB7061 did not itselfinhibit the growth of the target bacteria. Sensitization factor wasdefined as in Example 7. The results are shown in Table 9. NAB7061sensitized both carbapenem-resistant strains (F263, F264) to bothcarbapenems by a factor ≧4.

TABLE 9 Susceptibility of imipenem and meropenem against Acinetobacterbaumannii strains in the absence of NAB 7061 and in the presence of NAB7061 (4 μg/ml) MIC (μg/ml) of MIC (μg/ml) of imipenem at the meropenemat the indicated concn indicated concn (μg/ml) of (μg/ml) of NAB 7061NAB 7061 Strain 0 4 0 4 A. baumannii ATCC19606 0.38 0.38 1.5 0.75 A.baumannii F263 >32 8 >32 6 A. baumannii F264 24 6 32 4

Example 10 NAB7061 Sensitizes E. coli to the Complement in Fresh NormalSerum

The ability of NAB7061 to sensitize encapsulated, smooth strain of E.coli to the bactericidal action of normal guinea pig serum (GPS) wasstudied by the method described by Vaara et al. (1984). E. coli IH3080(018,K1) was grown in LB broth (LB broth Lennox, Difco, BD, Sparks, Md.,U.S.A) at 37° C. in a rotary shaker into early logaritmic growth phase,washed with PBS (phosphate-buffered saline, 8.0 g of NaCl, 0.2 g of KCl,1.44 g of Na₂HPO₄×2H₂O and 0.2 g of KH₂PO₄ per liter) and resuspended inPBS, to approx. 10⁹ cells/ml). GPS was used as complement source. It wasstored at −70° C. before use. To inactive the complement, serum wasincubated at 56° C. for 30 min.

The experimental procedure was as follows. 10% GPS in PBS was inoculatedwith approx. 500 CFU (colony forming units) of bacteria per ml andpipetted in 0.2 ml aliquots into wells of microtiter plates. The wellsalready contained increasing amounts of NAB7061 in 0.020 ml of 0.9%NaCl. The plate was incubated at 37° C. for 2 h whereafter each well wasemptied onto LB plates. The plates were incubated overnight at 37° C.and the developed colonies were counted.

The results are shown in Table 10. NAB7061 itself did not significantlyreduce CFU count in the absence of GPS or in the presence ofheat-inactivated 10% GPS. However, as low a concentration of NAB7061 as2 μg/ml was sufficient to reduce CFU count by a factor of approx. 100 inthe presence 10% fresh GPS. Accordingly, NAB7061 acts synergisticallywith the bactericidal complement machinery present in fresh serum, asdoes PMBN, the agent well known to have this property.

TABLE 10 The synergistic bactericidal activity of NAB7061 and 10% guineapig serum (GPS) against E. coli IH3080 (O18:K1)* Concentration ofNAB7061 (μg/ml) 0 1 2 4 none (PBS) 100 97 97 79 10% GPS 270 230 2 0 10%GPS, heat inactivated 500 500 500 250 *measured as % survival after2-hour treatment at 37° C.

Example 11 Reduced Affinity of NAB7061 to the Brush-Border Membrane(BBM) of the Renal Cortex

The binding of the compounds according to this invention to isolatedbrush-border membrane (BBM) from the renal cortex can be measuredindirectly by measuring their ability to inhibit the binding ofradiolabelled gentamycin to BBM. Accordingly, the compounds according tothis invention that have less affinity to BBM than for example polymyxinB inhibit the binding of radio-labelled gentamycin to a lesser degreethan does polymyxin B.

BBM was isolated from the renal cortex of male albino rats by using theMg²⁺/EGTA precipitation technique as described by Nagai et al. (2006).The binding of gentamycin was measured, according to the methoddescribed by Nagai et al. (2006) by incubating BBM vesicles (20 μl) in10 mM HEPES (pH 7.5) with 100 mM mannitol in the presence of 20 μM [³H]gentamycin (Amersham Biosciences Inc., Buckinghamshire, U.K.) with orwithout the compound to be tested or a positive control. After anincubation of 60 min at 4° C., 1 ml of ice-cold buffer described abovewas added, and the mixture was filtered through a Millipore filter (0.45μm; HAWP). The filter was washed with the buffer and the radioactivityremaining in the filter was measured by using a liquid scintillationcounter. The IC₅₀ values were determined as in Nagai et al. (2006) usingthe Hill equation.

The IC₅₀ values (μM) for the NAB compound studied and the controls werethe following: 187.3+24.3 for NAB7061 (average of two independentexperiments, each with three parallel determinations), 39.3+5.5 forpolymyxin B (average of two independent experiments, each with threeparallel determinations), and 90.2+9.7 for unlabelled gentamycin (threeparallel determinations). Accordingly, the affinity of NAB7061 to BBM isonly approximately half of the affinity of gentamycin to BBM andapproximately one fifth of the affinity of polymyxin B to BBM.

Example 12 The Activity of NAB7061 in Experimental E. coli PeritonitisModel in Mice

A suspension of E. coli IH3080 (K1:O18) in saline (0.9% NaCl) wasprepared from an overnight culture on a blood agar plate (Statens SerumInstitut, Copenhagen, Denmark). All mice (female NMR1 from HarlanScandinavia, Allerød, Denmark; weight, 25-30 g) were inoculatedintraperitoneally with 0.5 ml of the suspension containing 0.96×10⁶ CFUper ml in the lateral lower quadrant of the abdomen. At 1 h, the CFUcount was determined from three mice and the remaining mice (four miceper group) were treated with a subcutaneous injection of 0.2 ml oferythromycin solution in saline (corresponding to 5 mg/kg body weight)or NAB7061 solution in saline (corresponding to 5 mg/kg body weight), orboth erythromycin and NAB7061 (corresponding to 5 mg/kg body weight ofboth drugs; given at two separate sites). Control group received two 0.2ml injections of saline. At 4.5 h postinfection, all mice wereanaesthetized with CO₂ and sacrificed. Sterile saline (2 ml) wasinjected intraperitoneally and the abdomen was gently massaged before itwas opened and the fluid sampled. Appropriate dilutions of the fluidwere plated on blood agar plates, the plates were incubated overnight,and the colonies were counted.

At 1 h postinfection, the CFU count was 0.74 (+0.7)×10⁶ per ml. At 4.5 hpostinfection (corresponding to 3.5 h after the treatment), the CFUcounts (per ml) were 11.1 (±6.2)×10⁶ (control group), 8.9 (±6.4)×10⁶(erythromycin group), 1.1 (±0.6)×10⁶ (NAB 7061 group), and 2.1(±1.2)×10⁶ (NAB plus erythromycin group). Accordingly, in the absence ofNAB 7061, the bacterial count increased by a factor of 15 (saline group)or by a factor of 12 (erythromycin group), while in the presence of NAB7061, the corresponding factors ranged from 1.5 to 3.

Example 13 Toxicity Studies on NAB7061

Toxicity in young rats (weighing approx. 150 g in the beginning of thestudy) was determined by administering doses (1, 2, 4, 8, 16, and 32mg/kg per day) of NAB7061 as well as the control compound polymyxin Bintravenously twice (2) a day for two weeks. A group of ten (10) ratswas studied for each dosis regimen. Clinical observations were madedaily, the body weight measured twice weekly, and the food consumptiontwice weekly. In the end of week 2, all animals were sacrificed.

The control compound, polymyxin B negatively influenced the body weightgain at as low a dose as 1 mg/kg per day whereas the lowest dose ofNAB7061 that had this effect was 8 mg/kg. Polymyxin B caused mortalityat 32 mg/kg per day (100% mortality), whereas all rats receiving NAB7061remained alive throughout the study. In the end of the study, blood ureanitrogen (BUN) was 15% higher in the group receiving polymyxin 16 mg/kgper day than in the control or low dose polymyxin group (1 mg/kg perday). In the group that received NAB7061 at the dose of 16 mg/kg perday, no such a rise was found, and in the group that received NAB7061 atthe dose of 32 mg/kg per day, the rise was 7%.

Histopathology of kidneys was performed for each animals andhistopathology of all tissues was performed in three high-dose groupsreceiving NAB7061. No NAB7061-related pathological findings were foundin the general pathology and histopathology of any organs or in thehistopathology of kidneys in any animals that received NAB7061.

1. A polymyxin derivative of the general formula (I),

wherein R1, R2 and R3 are optional; and each represent any amino acidresidue, if present; R4 is an amino acid residue comprising a functionalside chain able to cyclicize said derivative; R6 is an independentlyselected optionally substituted hydrophobic amino acid residue; R7 is anindependently selected optionally substituted hydrophobic amino acidresidue or Thr; R5, R8 and R9 are cationic or neutral amino acidresidues; R10 is Leu, Thr or any non-hydrophobic amino acid residue; andR(FA) is an optionally substituted fatty acyl or alkyl residue, or ahydrophobic oligopeptide, wherein the alkyl residue has more than fivecarbon atoms; with the proviso that R8 and R9 are not both formylatedwhen R(FA)-R1—R2—R3 constitutes the native polymyxin B side chain; andR4 is not directly linked to octanoyl residue when R4—R10 constitutes anative polymyxin B ring structure; wherein said amino acid residues areselected so that the total number of positive charges at physiologicalpH is at least two but no more than three, and the total number ofpositive charges in the side chain portion, R(FA)-R1—R2—R3, does notexceed two, whereby said polymyxin derivative still possessesantibacterial activity against Gram-negative bacteria, and/or possessesthe ability to sensitize Gram-negative bacteria to antibacterial agents;or a pharmaceutically acceptable salt of said derivative.
 2. Thederivative according to claim 1, wherein said positive charges areselected from the group consisting of free, unsubstituted amino groupsand other cationic groups.
 3. The derivative according to claim 1,wherein R1—R10 is selected from the group consisting of SEQ ID NO: 9-26.4. The derivative according to claim 1, wherein R(FA) is selected fromthe group consisting of octanoyl, nonanoyl, isononanoyl, decanoyl,isodecanoyl, undecanoyl, dodecanoyl, tetradecanoyl, cyclohexanoyl,cycloheptanoyl, cyclooctanoyl, cyclononanoyl, cycloisononanoyl,cyclodecanoyl, cycloisodecanoyl, cycloundecanoyl, cyclododecanoyl,cyclotetradecanoyl, hexanoyl, heptanoyl, and 9-fluorenylmethoxy-carbonylresidues.
 5. The derivative according to claim 4, wherein R(FA) isselected from the group consisting of OA, DA and MHA.
 6. The derivativeaccording to claim 1, wherein the number of said positive charges isthree.
 7. The derivative according to claim 6, wherein R1-R10 isselected from the group consisting of SEQ ID NO: 9-20.
 8. The derivativeaccording to claim 7, selected from the group consisting of OA-SEQ IDNO. 10, DA-SEQ ID NO. 10, OA-SEQ ID NO. 11, OA-SEQ ID NO. 12, DA-SEQ IDNO. 13, OA-SEQ ID NO. 13, MHA-SEQ ID NO. 13, MHA-SEQ ID NO. 14, OA-SEQID NO. 15, OA-SEQ ID NO. 16, OA-SEQ ID NO. 17, OA-SEQ ID NO. 18, OA-SEQID NO. 19, OA-SEQ ID NO. 20, and DA-SEQ ID NO.
 9. 9. A combinationproduct comprising two or more different derivatives according toclaim
 1. 10. A pharmaceutical composition comprising at least onederivative according to claim 1, and at least one pharmaceuticallyacceptable carrier and/or excipient.
 11. The pharmaceutical compositionaccording to claim 10, further comprising an antibacterial agent.
 12. Amethod for developing antibiotics comprising the steps of a) providing anatural polymyxin or octapeptin compound, or a derivative thereof,having a total of 4 to 6 positive charges; b) substituting from 1 to 4residues carrying one or more positive charges with a residue not havinga positive charge, or with a covalent bond, thereby generating aderivative of a polymyxin compound having 2 or 3 positive charges; c)assaying said derivative compound for antibacterial activity againstGram-negative bacteria; or the ability to sensitize Gram-negativebacteria to an antibiotic; and d) selecting compounds havingantibacterial activity against Gram-negative bacteria, or the ability tosensitize Gram-negative bacteria to an antibacterial agent.
 13. Themethod according to claim 12 wherein said method is carried out as atotal synthetic process.
 14. The method according to claim 12, whereinsaid method is carried out as a semisynthetic process.
 15. A method forreducing the toxicity of natural polymyxins, octapeptins and theirderivatives for use in the treatment of infections in an individual,said method comprising the steps of: a) providing a natural polymyxin oroctapeptin compound, or a derivative thereof, having a total of 4 to 6positive charges; b) substituting from 1 to 4 residues carrying one ormore positive charges with a residue not having a positive charge, orwith a covalent bond, thereby generating a derivative having 2 or 3positive charges.
 16. The method according to claim 15, wherein saidmethod is carried out as a total synthetic process.
 17. The methodaccording to claim 15, wherein said method is carried out as asemisynthetic process.
 18. The method according to claim 15, whereinsaid toxicity is nephrotoxicity.
 19. A method for improving thepharmacokinetic properties, of natural polymyxins, octapeptins and theirderivatives, said method comprising the steps of: a) providing a naturalpolymyxin or octapeptin compound, or a derivative thereof, having atotal of 4 to 6 positive charges; b) substituting from 1 to 4 residuescarrying one or more positive charges with a residue not having apositive charge, or with a covalent bond, thereby generating aderivative having 2 or 3 positive charges; wherein said improvedpharmacokinetic properties consist of prolonged serum half life; orlower susceptibility to the inactivation by polyanionic tissue and pusconstituents, as compared to the starting compound.
 20. The methodaccording to claim 19, wherein said method is carried out as a totalsynthetic process.
 21. The method according to claim 19, wherein saidmethod is carried out as a semisynthetic process.
 22. A method forsensitizing clinically important Gram-negative bacteria to a hostdefense mechanism complement present in the serum, wherein said bacteriaare subjected to the action of a derivative according to claim 1 duringa clinical infection.
 23. The method according to claim 22, wherein saidbacteria are selected from the group consisting of: Escherichia coli,Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae,Citrobacter freundii, Pseudomonas aeruginosa, and Acinetobacterbaumannii.
 24. A method of treating infections caused by Gram-negativebacteria comprising administering a derivative according to claim
 1. 25.The method according to claim 24, wherein said bacteria are selectedfrom the group consisting of: Escherichia coli, Klebsiella pneumoniae,Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii,Pseudomonas aeruginosa, and Acinetobacter baumannii.
 26. A method ofsensitizing Gram-negative bacteria against anti-bacterial agentscomprising administering a derivative according to claim
 1. 27. Themethod of claim 26, wherein said bacteria are selected from the groupconsisting of: Escherichia coli, Klebsiella pneumoniae, Klebsiellaoxytoca, Enterobacter cloacae, Citrobacter freundii, Pseudomonasaeruginosa, and Acinetobacter baumannii.
 28. The method of claim 26,wherein said antibacterial agent is selected from the group consistingof clarithromycin, azithromycin, erythromycin and other macrolides,ketolides, clindamycin and other lincosamines, streptogramins, rifampin,rifabutin, rifalazile and other rifamycins, fusidic acid, mupirocin,oxazolidinones, vancomycin, dalbavancin, telavancin, oritavancin andother glycopeptide antibiotics, fluoroquinolones, bacitracin,tetracycline derivatives, betalactam antibiotics, novobiocin,pleuromutilins, folate synthesis inhibitors, deformylase inhibitors, andbacterial efflux pump inhibitors.
 29. The method of claim 28, whereinsaid antibacterial agent is selected from the group consisting of:clarithromycin, azithromycin, erythromycin, clindamycin, thestreptogramin combination quinupristin-dalfopristin, rifampin, fusidicacid, mupirocin, the oxazolidinone linezolid, vancomycin, thefluoroquinolone moxifloxacin, and the folate synthesis inhibitortrimetoprim.
 30. A method for sensitizing Gram-negative bacteria to ahost defense mechanism complement present in the serum comprising theuse of a derivative according to claim
 1. 31. The method according toclaim 30, wherein said bacteria are selected from the group consistingof: Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca,Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, andAcinetobacter baumannii.
 32. A process for preparing a polymyxinderivative of formula (I) as defined in claim 1 comprising modifying anatural or synthetic polymyxin or octapeptin compound or a derivativethereof having 4 to 6 positively charged residues by replacing 1 to 4 ofsaid residues by neutral residues, or by a covalent bond, or converting1 to 4 of said residues into neutral residues in order to obtain apolymyxin derivative of formula (I) according to claim 1, having 2 or 3positively charged residues.
 33. The process according to claim 32comprising carrying out the process as a total synthetic process. 34.The process according to claim 32 comprising carrying out the process asa semisynthetic process.
 35. The process according to claim 32comprising the steps of: a) subjecting a natural or synthetic polymyxinor octapeptin compound or a derivative thereof to cleavage in order toremove the side chain of said polymyxin compound, and recovering thecyclic part of said compound, and b) coupling to the cyclic partobtained in step a) a synthetically prepared side chain in order toobtain a polymyxin derivative of formula (I) according to claim
 1. 36.The process according to claim 35 comprising carrying out the cleavagein step a) enzymatically.
 37. The process according to claim 35comprising carrying out the cleavage in step a) chemically.
 38. Theprocess according to claim 35 comprising carrying out the cleavage instep a) using a combination of both chemical and enzymatic treatments.